DNA molecules encoding L-glutamate-gated chloride channels from Schistocerca americana

ABSTRACT

The present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode  Schistocerca americana  (grasshopper) glutamate-gated chloride channels. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding  S. americana  glutamate-gated chloride channels, substantially purified forms of associated  S. americana  glutamate-gated chloride channels and recombinant membrane fractions comprising these proteins, associated mutant proteins, and methods associated with identifying compounds which modulate associated  Schistocerca americana  glutamate-gated chloride channels, which will be useful as insecticides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/160,877, filed Oct. 22, 1999, under 35 U.S.C. § 119(e).

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not Applicable

REFERENCE TO MICROFICHE APPENDIX

Not Applicable

FIELD OF THE INVENTION

The present invention relates in part to isolated nucleic acid molecules(polynucleotides) which encode Schistocerca americana (grasshopper)glutamate-gated chloride channels. The present invention also relates torecombinant vectors and recombinant hosts which contain a DNA fragmentencoding S. americana glutamate-gated chloride channels, substantiallypurified forms of associated S. americana glutamate-gated chloridechannels and recombinant membrane fractions comprising these proteins,associated mutant proteins, and methods associated with identifyingcompounds which modulate associated Schistocerca americanaglutamate-gated chloride channels, which will be useful as insecticides.

BACKGROUND OF THE INVENTION

Dudel et al. (1989, Brain Res. 481: 215–220) demonstrated direct gatingof an ion channel by glutamate in the arthropod, Schistocerca gregaria(locust) leg muscle.

Glutamate-gated chloride channels, or H-receptors, have been identifiedin arthropod nerve and muscle (Lingle et al, 1981, Brain Res. 212:481–488; Horseman et al., 1988, Neurosci. Lett. 85: 65–70; Wafford andSattelle, 1989, J. Exp. Bio. 144: 449–462; Lea and Usherwood, 1973,Comp. Gen. Parmacol. 4: 333–350; and Cull-Candy, 1976, J. Physiol. 255:449–464).

Additionally, glutamate-gated chloride channels have been cloned fromthe soil nematode Caenorhabditis elegans (Cully et al., 1994, Nature371: 707–711; see also U.S. Pat. No. 5,527,703 and Arena et al., 1992,Molecular Brain Research. 15: 339–348) and Drosophila melanogaster(Cully et al., 1996, J. Biol. Chem. 271: 20187–20191).

Raymond et al. (2000, Neuroreport 11: 2695–2701) disclose detection ofGluCl channels in dorsal median neurons in Periplanta americana(cockroach).

Invertebrate glutamate-gated chloride channels are important targets forthe widely used avermectin class of anthelmintic and insecticidalcompounds. The avermectins are a family of macrocyclic lactonesoriginally isolated from the actinomycete Streptomyces avermitilis. Thesemisynthetic avermectin derivative, ivermectin(22,23-dihydro-avermectin B_(1a)), is used throughout the world to treatparasitic helminths and insect pests of man and animals. The avermectinsremain the most potent broad spectrum endectocides exhibiting lowtoxicity to the host. After many years of use in the field, thereremains little resistance to avermectin in the insect population. Thecombination of good therapeutic index and low resistance stronglysuggests that the glutamate-gated chloride (GluCl) channels remain goodtargets for insecticide development.

Despite the identification of the aforementioned cDNA clones encodingGluCl channels, it would be advantageous to identify additionalinvertebrate genes encoding GluCl channels in order to allow screeningto identify novel GluCl channel modulators that may have insecticidal,mitacidal and/or nematocidal activity for animal health or cropprotection. The present invention addresses and meets these needs bydisclosing isolated nucleic acid molecules which express a Schistocercaamericana GluGl channel wherein expression of grasshopper GluCl cRNA inXenopus oocytes or other appropriate host cell results in an activeGluCl channel. Heterologous expression of a Schistocerca americana(grasshopper) GluCl channels will allow the pharmacological analysis ofcompounds active against parasitic invertebrate species relevant toanimal and human health. Such species include worms, fleas, tick, andlice. Heterologous cell lines expressing an active GluCl channel can beused to establish functional or binding assays to identify novel GluClchannel modulators that may be useful in control of the aforementionedspecies groups.

SUMMARY OF THE INVENTION

The present invention relates to an isolated or purified nucleic acidmolecule (polynucleotide) which encodes a novel Schistocerca americana(grasshopper) invertebrate GluCl channel protein.

The present invention relates to an isolated of purified nucleic acidmolecule (polynucleotide) which encodes mRNA which expresses a novelSchistocerca americana (grasshopper) invertebrate GluCl channel protein,this DNA molecule comprising the nucleotide sequence disclosed herein asSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9.

The present invention also relates to biologically active fragments ormutants of SEQ ID NOs:1, 3, 5, 7 and 9 which encodes mRNA expressing anovel Schistocerca americana (grasshopper) invertebrate GluCl channelprotein. Any such biologically active fragment and/or mutant will encodeeither a protein or protein fragment which at least substantially mimicsthe pharmacological properties of a grasshopper GluCl channel protein,including but not limited to the grasshopper GluCl channel proteins asset forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQID NO:10. Any such polynucleotide includes but is not necessarilylimited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a functional S. americanaGluCl channel in a eukaryotic cell, such as Xenopus oocytes, so as to beuseful for screening for agonists and/or antagonists of S. americanaGluCl activity.

A preferred aspect of this portion of the present invention is disclosedin FIGS. 1A–F, cDNA molecule (SEQ ID NOs:1, 3, 5, 5 and 9) encoding anovel Schistocerca americana (grasshopper) GluCl protein.

The isolated nucleic acid molecules of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification.

The present invention also relates to subcellular membrane fractions ofthe recombinant host cells (both prokaryotic and eukaryotic as well asboth stably and transiently transformed cells) which contain thefunctional and processed proteins encoded by the nucleic acids of thepresent invention. The present invention relates to a substantiallypurified membrane preparation which comprises a S. americana GluClchannel and is essentially free from contaminating proteins, includingbut not limited to other S. americana source proteins or host proteinsfrom a recombinant cell which expresses SaGluCl1 or SaGluCl2. Especiallypreferred is a membrane preparation which contains a S. americana GluClchannel comprising a GluCl protein comprising the functional form of thefull length GluCl channel proteins as disclosed in FIGS. 2A–B and as setforth in SEQ ID NOs: 2, 4, 6, 8 and 10. To this end, the presentinvention also relates to a substantially purified membrane preparationwhich is purified from a recombinant host, whether a recombinanteukaryotic or recombinant prokaryotic host, wherein a recombinant vectorexpresses a functional and fully processed S. americana GluCl channel.Especially preferred is a membrane preparation which comprises arecombinant form of the S. americana GluCl channel, SaGluCl1, SaGluCl2or any heteromultimer combination wherein a protein sequence comprises afunctional portion of the full length SaGluCl proteins as disclosed inFIGS. 2A–B and as set forth in SEQ ID NOs: 2, 4, 6, 8 and 10. Thesesubcellular membrane fractions will comprise either wild-type or mutantvariations which are biologically functional forms of the S. americanaGluCl channel, SaGluCl1, SaGluCl2 or any heteromultimer combinationthereof at levels substantially above endogenous levels and hence willbe useful in various assays described throughout this specification. Apreferred eukaryotic host of choice to express the glutamate-gatedchannels of the present invention are Xenopus oocytes.

It is exemplified herein that expression of either DNA molecule encodinga short form of SaGluCl1 (i.e., SEQ ID NO:5 and/or 7) in Xenopus oocytesof chinese hamster ovary (CHO) cells results in a functional GluClchannel. In contrast, co-expression of SaGluCl2 (from, e.g., SEQ IDNO:9) with a short form of SaGluCl1 prevents the formation of afunctional channel otherwise detectable when the short form of SaGluCl1is expressed alone. Expression of either long form of SaGluCl1 is shownto be non-functional to date in Xenopus oocytes. The short formexemplified herein is expression of the “SC” form (SEQ ID NO:7,expression the precursor protein containing the amino acid sequence asset forth in SEQ ID NO:8, the expression in Xenopus oocytes or CHO cellsresulting in formation of the functional GluCl channel).

The present invention also relates to a substantially purified form ofan S. americana GluCl channel protein, which comprises the amino acidsequence disclosed in FIGS. 2A–B and set forth as SEQ ID NOs:2, 4, 6, 8and 10.

A preferred aspect of this portion of the present invention is a an S.americana GluCl channel protein which consists of the amino acidsequence disclosed in FIGS. 2A–B and set forth as SEQ ID NOs:2, 4, 6, 8and 10.

Another preferred aspect of the present invention relates to asubstantially purified, fully processed GluCl channel protein obtainedfrom a recombinant host cell containing a DNA expression vectorcomprises a nucleotide sequence as set forth in SEQ ID NOs: 1, 3, 5, 7,and/or 9 and expresses the respective SaGluCl precursor protein. It isespecially preferred is that the recombinant host cell be a eukaryotichost cell, such as a mammalian cell line, or Xenopus oocytes, as notedabove.

Another preferred aspect of the present invention relates to asubstantially purified membrane preparation which has been obtained froma recombinant host cell transformed or transfected with a DNA expressionvector which comprises and appropriately expresses a complete openreading frame as set forth in SEQ ID NOs: 1, 3, 5, 7, and/or 9,resulting in a functional, processed form of the respective SaGluClchannel. It is especially preferred is that the recombinant host cell bea eukaryotic host cell, such as a mammalian cell line, or Xenopusoocytes, as noted above.

The present invention also relates to biologically active fragmentsand/or mutants of an S. americana GluCl channel protein, comprising theamino acid sequence as set forth in SEQ ID NOs:2, 4, 6, 8 and/or 10,including but not necessarily limited to amino acid substitutions,deletions, additions, amino terminal truncations and carboxy-terminaltruncations such that these mutations provide for proteins or proteinfragments of diagnostic, therapeutic or prophylactic use and would beuseful for screening for selective modulators, including but not limitedto agonists and/or antagonists for S. americana GluCl channelpharmacology.

A preferred aspect of the present invention is disclosed in FIGS. 2A–Band is set forth as SEQ ID NOs:2, 4, 6, 8 and 10, respective amino acidsequences which encode grasshopper GluCl proteins. Characterization ofone or more of these channel proteins allows for screening methods toidentify novel GluCl channel modulators that may have insecticidal,mitacidal and/or nematocidal activity for animal health or cropprotection. As noted above, heterologous expression of a Schistocercaamericana (grasshopper) GluCl channels will allow the pharmacologicalanalysis of compounds active against parasitic invertebrate speciesrelevant to animal and health. Such species include worms, fleas, tick,and lice. Heterologous cell lines expressing a functional SaGluClchannel (e.g., functional forms of SEQ ID NO:6 and 8, including but notlimited to mature forms generated via heterologous expression of SEQ IDNO: 5 or 7) can be used to establish functional or binding assays toidentify novel GluCl channel modulators that may be useful in control ofthe aforementioned species groups. Additionally, co-expression of afunctional SaGluCl protein with SaGluCl2 results in SaGluCl2 imparting adominant negative effect on channel activity, which is also useful invarious assays utilized to identify modulators of an in vivo GluClchannel.

The present invention also relates to polyclonal and monoclonalantibodies raised in response to either the form of SaGluCl, or abiologically active fragment thereof.

The present invention also relates to SaGluCl1 and/or SaGlu2 fusionconstructs, including but not limited to fusion constructs which expressa portion of the SaGluCl1 and/or SaGlu2 linked to various markers,including but in no way limited to GFP (Green fluorescent protein), theMYC epitope, and GST. Any such fusion constructs may be expressed in thecell line of interest and used to screen for modulators of one or moreof the SaGluCl proteins disclosed herein.

The present invention relates to methods of expressing grasshopper GluClchannel proteins biological equivalents disclosed herein, assaysemploying these gene products, recombinant host cells which comprise DNAconstructs which express these proteins, and compounds identifiedthrough these assays which act as agonists or antagonists of GluClchannel activity.

It is an object of the present invention to provide an isolated nucleicacid molecule (e.g., SEQ ID NOs:1, 3, 5, 7, and 9) which encodes a novelform of grasshopper GluCl, or fragments, mutants or derivatives of SEQID NOs:2, 4, 6, 8 and 10. Any such polynucleotide includes but is notnecessarily limited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a protein or protein fragmentof diagnostic, therapeutic or prophylactic use and would be useful forscreening for selective modulators for invertebrate GluCl pharmacology.

It is a further object of the present invention to provide thegrasshopper GluCl proteins or protein fragments encoded by the nucleicacid molecules referred to in the preceding paragraph.

It is a further object of the present invention to provide recombinantvectors and recombinant host cells which comprise a nucleic acidsequence encoding grasshopper GluCl proteins or a biological equivalentthereof.

It is an object of the present invention to provide a substantiallypurified form of grasshopper GluCl proteins, as set forth in SEQ IDNOs:2, 4, 6, 8, and 10.

Is is another object of the present invention to provide a substantiallypurified recombinant form of a grasshopper GluCl protein which has beenobtained from a recombinant host cell transformed or transfected with aDNA expression vector which comprises and appropriately expresses acomplete open reading frame as set forth in SEQ ID NOs: 1, 3, 5, 7,and/or 9, resulting in a functional, processed form of the respectiveSaGluCl channel. It is especially preferred is that the recombinant hostcell be a eukaryotic host cell, such as a mammalian cell line.

It is an object of the present invention to provide for biologicallyactive fragments and/or mutants of grasshopper GluCl proteins, such asset forth in SEQ ID NOs:2, 4, 6, 8 and 10, including but not necessarilylimited to amino acid substitutions, deletions, additions, aminoterminal truncations and carboxy-terminal truncations such that thesemutations provide for proteins or protein fragments of diagnostic,therapeutic and/or prophylactic use.

It is further an object of the present invention to provide forsubstantially purified subcellular membrane preparations which comprisepharmacologically active grasshopper GluCl channels, especiallysubcellular fractions obtained from a host cell transfected ortransformed with a DNA vector comprising a nucleotide sequence whichencodes a protein which comprises the amino acid as set forth in SEQ IDNO:2, 4, 6, 8 and 10 and FIGS. 2A–B.

Is is another object of the present invention to provide a substantiallypurified membrane preparations obtained from a recombinant host celltransformed or transfected with a DNA expression vector which comprisesand appropriately expresses a complete open reading frame as set forthin SEQ ID NOs: 1, 3, 5, 7, and/or 9, resulting in a functional,processed form of the respective SaGluCl channel. It is especiallypreferred is that the recombinant host cell be a eukaryotic host cell,such as a mammalian cell line, or Xenopus oocytes.

It is also an object of the present invention to use grasshopper GluClproteins or membrane preparations containing grasshopper GluCl proteinsor a biological equivalent to screen for modulators, preferablyselective modulators, of grasshopper GluCl channel activity. Any suchcompound may be useful in screening for and selecting compounds activeagainst parasitic invertebrate species relevant to animal and health.Such species include worms, fleas, tick, and lice. These membranepreparations may be generated from heterologous cell lines expressingthese GluCl and may constitute full length protein, biologically activefragments of the full length protein or may rely on fusion proteinsexpressed from various fusion constructs which may be constructed withmaterials available in the art.

As used herein, “substantially free from other nucleic acids” means atleast 90%, preferably 95%, more preferably 99%, and even more preferably99.9%, free of other nucleic acids. Thus, a grasshopper GluCl DNApreparation that is substantially free from other nucleic acids willcontain, as a percent of its total nucleic acid, no more than 10%,preferably no more than 5%, more preferably no more than 1%, and evenmore preferably no more than 0.1%, of non-grasshopper GluCl nucleicacids. Whether a given grasshopper GluCl DNA preparation issubstantially free from other nucleic acids can be determined by suchconventional techniques of assessing nucleic acid purity as, e.g.,agarose gel electrophoresis combined with appropriate staining methods,e.g., ethidium bromide staining, or by sequencing.

As used herein, “substantially free from other proteins” or“substantially purified” means at least 90%, preferably 95%, morepreferably 99%, and even more preferably 99.9%, free of other proteins.Thus, an grasshopper GluCl protein preparation that is substantiallyfree from other proteins will contain, as a percent of its totalprotein, no more than 10%, preferably no more than 5%, more preferablyno more than 1%, and even more preferably no more than 0.1%, ofnon-grasshopper GluCl proteins. Whether a given grasshopper GluClprotein preparation is substantially free from other proteins can bedetermined by such conventional techniques of assessing protein purityas, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) combined with appropriate detection methods, e.g., silverstaining or immunoblotting. As used interchangeably with the terms“substantially free from other proteins” or “substantially purified”,the terms “isolated grasshopper GluCl protein” or “purified grasshopperGluCl protein” also refer to grasshopper GluCl protein that has beenisolated from a natural source. Use of the term “isolated” or “purified”indicates that grasshopper GluCl protein has been removed from itsnormal cellular environment. Thus, an isolated grasshopper GluCl proteinmay be in a cell-free solution or placed in a different cellularenvironment from that in which it occurs naturally. The term isolateddoes not imply that an isolated grasshopper GluCl protein is the onlyprotein present, but instead means that an isolated grasshopper GluClprotein is substantially free of other proteins and non-amino acidmaterial (e.g., nucleic acids, lipids, carbohydrates) naturallyassociated with the grasshopper GluCl protein in vivo. Thus, agrasshopper GluCl protein that is recombinantly expressed in aprokaryotic or eukaryotic cell and substantially purified from this hostcell which does not naturally (i.e., without intervention) express thisGluCl protein is of course “isolated grasshopper GluCl protein” underany circumstances referred to herein. As noted above, a grasshopperGluCl protein preparation that is an isolated or purified grasshopperGluCl protein will be substantially free from other proteins willcontain, as a percent of its total protein, no more than 10%, preferablyno more than 5%, more preferably no more than 1%, and even morepreferably no more than 0.1%, of non-grasshopper GluCl proteins.

As used interchangeably herein, “functional equivalent” or “biologicallyactive equivalent” means a protein which does not have exactly the sameamino acid sequence as naturally occurring grasshopper GluCl, due toalternative splicing, deletions, mutations, substitutions, or additions,but retains substantially the same biological activity as grasshopperGluCl. Such functional equivalents will have significant amino acidsequence identity with naturally occurring grasshopper GluCl and genesand cDNA encoding such functional equivalents can be detected by reducedstringency hybridization with a DNA sequence encoding naturallyoccurring Grasshopper GluCl. For example, a naturally occurringgrasshopper GluCl disclosed herein comprises the amino acid sequenceshown as SEQ ID NO:2 and is encoded by SEQ ID NO:1. A nucleic acidencoding a functional equivalent has at least about 50% identity at thenucleotide level to SEQ ID NO:1.

As used herein, “a conservative amino acid substitution” refers to thereplacement of one amino acid residue by another, chemically similar,amino acid residue. Examples of such conservative substitutions are:substitution of one hydrophobic residue (isoleucine, leucine, valine, ormethionine) for another; substitution of one polar residue for anotherpolar residue of the same charge (e.g., arginine for lysine; glutamicacid for aspartic acid).

As used herein, “GluCl” refers to—L-glutamate-gated chloride channel—.

As used herein, the term “mammalian” will refer to any mammal, includinga human being.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–F compares the nucleotide sequence of the DNA molecules whichencode respective grasshopper GluCl proteins, as set forth in SEQ IDNOs:1, 3, 5, 7, and 9.

FIGS. 2A–B show the amino acid sequence of the grasshopper GluClproteins as set forth in SEQ ID NO:2, 4, 6, 8, and 10.

FIG. 3 shows the results of an experiment in which the clone SaGluCl1(short form “SC”) was expressed in a Xenopus oocyte. The measurement ismade with the two microelectrode voltage clamp technique and themembrane potential was held at 0 mV. Two current recordings aresuperimposed. The bars at top show the duration of application ofglutamate and ivermectin phosphate.

FIG. 4A and FIG. 4B show the SaGluCl1 (short form “SC”) expressed in CHOcells assembles into a homomultimer and is activated by ivermectin (FIG.4A) and nodulasporic acid (FIG. 4B). 100 nM ivermectin and 1 μMnodulasporic acid were added at 20 seconds and current was measured.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes a Schistocerca americana (grasshopper)invertebrate GluCl channel protein. The nucleic acid molecules of thepresent invention are substantially free from other nucleic acids. Formost cloning purposes, DNA is a preferred nucleic acid.

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes mRNA which expresses a novel Schistocercaamericana (grasshopper) invertebrate GluCl channel protein, this DNAmolecule comprising the nucleotide sequence disclosed herein as SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9.

The isolation and characterization of the SaGluCl nucleic acid moleculesof the present invention were identified through degenerate PCR asdescribed in detail in Example Section 1. PCR products of theappropriate size were cloned into the PCR2.1 plasmid vector using a “TA”cloning kit (Invitrogen, Inc.). Following sequence analysis (ABI Prism,PE Applied Biosystems), selected PCR clone inserts were radiolabelledand used as probes to screen a cDNA library generated into the Uni-ZAPvector (Stratagene, Inc.) from using and a poly (A) enriched fractionfrom the RNA mentioned above. Sequences from full-length cDNA cloneswere analyzed using the GCG Inc. package. Subcloning of SaGluCl1 clonesand of SaGluCl2 into a mammalian expression vector was done by excisionof whole inserts (EcoRI+XhoI excision) from the UniZap pBS plasmid,followed by ligation into the TetSplice (Life Technologies Inc.).Invertebrate glutamate-gated chloride channels (GluCls) are related tothe glycine- and GABA-gated chloride channels and are distinct from theexcitatory glutamate receptors (e.g. NMDA or AMPA receptors). The firsttwo members of the GluCl family were identified in the nematode C.elegans, following a functional screen for the receptor of theanthelmintic drug ivermectin. Several additional GluCls have now beencloned in other invertebrate species. However, there is no evidence yetfor GluCl counterparts in vertebrates; because of this, GluCls areexcellent targets for anthelmintics, insecticides, acaricides, etc.Specific GluCl modulators, such as nodulasporic acid and itsderivatives, indeed have an ideal safety profile because they lackmechanism-based toxicity in vertebrates. The present invention relatesin part to two novel GluCl clones, SaGluCl1 and GluCl2, from the ventralganglia of the grasshopper Schistocerca americana byreverse-transcriptase polymerase chain reaction (RT-PCR) usingdegenerate oligonucleotides as reaction primers. Sequence data offull-length SaGluCl1 cDNA clones (SEQ ID NOs: 1, 3, 5, and 7) showshomology to D. melanogaster DrosGluClα and to C. felis CfGluCl DNA.Heterologous expression of the short form of SaGluCl1 into Xenopuslaevis oocytes results in a robust L-glutamate-gated chloride current.This channel is also activated by nodulasporic acid and ivermectin.Sequence of full-length SaGluCl2 cDNA clone (SEQ ID NO;9) shows amoderate homology with various GluCls. Co-expression of SaGluCl2 withSaGluCl1 strongly reduces the response to the compounds mentioned above,suggesting a modulator role for SaGluCl2. From previous work on otherfour-transmembrane ligand-gated channels receptors (Acetylcholine, GABAreceptors, etc.) it emerges that most, if not all, native channels areheteromultimers, i.e. pentamers that include at least two differentsubunits. In an attempt to demonstrate that SaGluCl1 and SaGluCl2 canco-assemble into a functional channel, both clones were co-expressed inXenopus oocytes and CHO cells. No current was observed in either casewhen both clones were co-expressed. It therefore appears that, underthese heterologous expression conditions, SaGluCl2 has a dominantnegative effect on SaGluCl1. Since data nevertheless indicate that bothsubunits are highly expressed in the same neurons, a third component ofthe native GluCl possibly remains to be identified. Alternatively, thepossibility remains that SaGluCl1 and SaGluCl2 are not expressed withincommon (neuronal) cells within the ganglia and may not co-assemble intoa common, functional channel. Regardless, as shown in FIG. 3 (Xenopusoocytes) and FIGS. 4A and 4B (CHO cells), expression of the short formof SaGluCl1 results in formation of a functional homomultimer channel,responsive to glutamate, ivermectin and nodulasporic acid.

Therefore, SaGluCl2, when co-expressed with the short form of SaGluCl1,prevents the formation of a functional channel otherwise detectable withSaGluCl1 alone. The SaGluCl2 protein is therefore expressed, acting as amodulator of SaGluCl1. Both SaGluCl1 and SaGluCl2 are expressed in thesame tissue and this tissue shows GluCl activity as recordedelectrophysiologically. Therefore, either 1) SaGluCl1 and SaGluCl2 arenot expressed in the exact same cells and/or 2) there is an additional,yet to be identified factor which is either be another SaGluCl form, oran unrelated type of protein such as that seen with the TipE proteinfrom Drosophila, which enhances in vitro expression of the para sodiumchannel (see Feng et al., 1995, Cell 82:1001–1011). It has also beenshown that SaGluCl2 can negatively affect non-SaGluCl four-transmembraneligand-gated ion channels (4TMLGIC), but does not likely exert anyeffect on other type of ion channels. Therefore, the SaGluCl protein maybe classified as a potential broad spectrum inhibitory subunit as itrelates specifically to four-transmembrane ligand-gated ion channels,lending this isolated cDNA clones, associated vectors, hosts,recombinant subcellular fractions and membranes, and the expressed andmature forms of SaGluCl2 as important tools for drug discovery.

To this end, the present invention relates in part to transgenicanimals, either an invertebrate (e.g., C. elegans) or vertebrate (e.g.,mouse), for which the gene encoding SaGluCl2 has been introduced intothe germline of the animal. The purpose of this would be to inactivate,in the host, one or several endogenous 4TMLGIC and observe thebiological effects. One such effect may well be an acquired resistanceto drugs that are agonists (activators) of 4TMLGIC. In the case of drugswith suspected—but unproven—method of action (MOA) via 4TMLGIC, suchSaGluCl2-harboring transgenic animals may be used to confirm such aneffect. Expression of the newly introduced gene encoding SaGluCl2 intothe host can be constitutive or inducible, depending on the type ofpromoter used to drive its expression. Also depending on the type ofpromoter used, expression of SaGluCl2 can be targeted to a giventissue(s) or it can be generalized. The same logic can be applied to invitro expression in e.g. Xenopus oocytes. Co-expression of SaGluCl2 withany other 4TMLGIC (including functional SaGluCl1 forms as well asnon-locust forms) should prevent any 4TMLGIC-mediated current. Xenopusoocytes may be injected with mixed populations of RNA or mRNA from e.g.mammalian brain or from whole animals (e.g. C. elegans). These oocytesare then subjected to drugs of sometimes unknown MOA. The use ofSaGluCl2 may lead to inhibition of the drug response, hence narrowingdown the investigative process to 4TMLGICs and in turn helping toidentify preferred modulators of various 4TLMGICs.

Therefore, the heterologous expression of grasshopper GluCl channels,especially SaGluCl1 alone or the co-expression of SaGluCl2 as describedabove, will allow the pharmacological analysis of compounds activeagainst parasitic invertebrates species relevant to animal and humanhealth. Such species include worms, fleas, tick, and lice. Heterologouscell lines expressing these GluCl channels can be used to establishfunctional or binding assays to identify novel GluCl channel modulatorsthat may be useful in control of the aforementioned species groups.

The DNA molecule described in FIGS. 1A–F as SAGluCl1LA (Sa1LA) and setforth as SEQ ID NO:1, which encodes the grasshopper GluCl proteindescribed in FIGS. 2A–B as 1LA and set forth as SEQ ID NO:2, thenucleotide sequence as follows:

GCCGCACGTG CCGCACGCCT CGCCACGCCG CACCGCAGGT CTCTTCGCCC ATGGAGGAGCTCGTGCGACT GGAGTGAGCT TGTGGCTTAC GGCTCTATCC GAGGAACCAT AAGCAAGTCACCGGCAGTTG AGGGTTAACT GGTAATGCGG GCGCGGGTCG AGGCCAGCAG AGACCGCCGCGTGCCGCCCG CGGACTCGCA CTGCCCCCCG GACGCGCCGC CCCGGCCGCC AGCCGCGCGCTCGCCCTGCT CCCACCGCCT CTGAAGTTCG AGGGACAGCT CAGCCTCAGC ATCCCTACAGGAACCATGAT GAGCGACCTC TGCAAGACGC CGTGGTGGCT TTGTGCGCTG CTGTTGCTGGCCGCCAACGT GCCAGATGCC TGGTGCACGC AGCAGAAGAT GAACTTCCGC GAGAAGGAGAAACAGGTGCT GGACCAGATC CTGGGGCCAG GTCGCTACGA CGCGCGCATC CGGCCGTCGGGGATCAACGG CACAGCGGAC AACCCCACCA TAGTGAAAGT GAACATCTTC CTTCGCTCTATCAGCAAAAT AGACGATTAT AAAATGATGT TCCGCTGCGC TGCCCCGCTT GCATGCCGCCCGGACCCCGC ACGCCGCCCT CGGCCCAGCG TCATGTGTTT TGTTACACGT GTTCTCAACAGGAGCCAGGC TCACCGACCC GGCCGCCAAT CAGGCGCCGC GTGTGCGACT CCGAACAAGGAATACAGCGT CCAGCTGACA TTCAGAGAGC AGTGGATGGA TGAGCGGCTC AAGTTCAATGACTTCAAAGG AAAAATAAAG TACCTGACAC TGACAGACGC CAACAGAGTA TGGATGCCAGATTTATTTTT CTCTAACGAA AAGGAAGGAC ATTTTCATAA CATTATCATG CCCAACGTTTATATACGTAT TTTTCCTTAT GGATCGGTGC TCTACAGTAT CAGAATCTCT CTGACSCTCTCCTGCCCGAT GAACCTGAAG CTGTACCCGC TCGACAGGCA GGTCTGCTCG CTGAGGATGGCCAGCTATGG TTGGACGACT GATGACCTGG TGTTCCTGTG GAAAGACGGC GACCCGGTGCAGGTGGTCAA GAACCTGCAC CTGCCGCGCT TCACGCTAGA GAAGTTCCTT ACCGACTACTGCAACAGCAA AACTAACACA GGAGAGTACA GCTGCTTGAA GGTGGACCTG CTGTTCAACCGCGAGTTCAG CTACTACCTG ATCCAGATCT ACATCCCGTG CTGCATGCTG GTGATAGTGTCGTGGGTGTC CTTCTGGCTC GATCAGAGCG CCATCCCGGC ACGGGTGTCC CTAGGCGTGACCACGCTGCT CACCATGGCC ACCCAGACGT CGGGCATCAA CGCCTCCCTG CCCCCCGTGTCCTATACCAA AGCCATCGAC GTGTGGACTG GCGTGTGCCT CACTTTCGTG TTCGGCGCGTTGCTCGAGTT CGCGCTCGTC AACTACGCGA GCCGCTCGGA CATGCACCGC GAGAACATGAAGAAGCAGCG CCGCCAGGTA GAGCTGGAGC ACGCCGCGCA GCTCGAGGCG GCCGCTGACCTGCTCGTGGA GGACGGCAGC ACCACCTTTG CCATGAAGCC GTTGGTGGGA CACCCGGGCGGTCCGGCGGC CGCAGCGCCC GGCCTCGCAG GCCTGGACAA GGTGCGCCAG TGCGAGATCCACATGCAGCC CAAGCGCGAA AACTGCTGCC GCACCTGGCT CTCCAAGTTC CCCACGCGCTCCAAGCGCAT CGACGTCATC TCGCGCATCA CCTTCCCGCT CGTCTTCGCG CTCTTCAACCTCGTCTACTG GTCCACCTAC CTGTTCCGCG AGGACGAACG CGAGTGAGCC GCCAGCCGCCATCGCAGCAG CAGCCAGCT (SEQ ID NO:1).

The DNA molecule described in FIGS. 1A–F as SAGluCl1LC (Sa1LC) and setforth as SEQ ID NO:3, which encodes the grasshopper GluCl proteindescribed in FIGS. 2A–B as 1LC and set forth as SEQ ID NO:4, thenucleotide sequence as follows:

GCCGCACGTG CCGCACGCCT CGCCACGCCG CACCGCAGGT CTCTTCGCCC ATGGAGGAGCTCGTGCGACT GGAGTGAGCT TGTGGCTTAC GGCTCTATCC GAGGAACCAT AAGCAAGTCACCGGCAGTTG AGGGTTAACT GGTAATGCGG GCGCGGGTCG AGGCCAGCAG AGACCGCCGCGTGCCGCCCG CGGACTCGCA CTGCCCCCCG GACGCGCCGC CCCGGCCGCC AGCCGCGCGCTCGCCCTGCT CCCACCGCCT CTGAAGTTCG AGGGACAGCT CAGCCTCAGC ATCCCTACAGGAACCATGAT GAGCGACCTC TGCAAGACGC CGTGGTGGCT TTGTGCGCTG CTGTTGCTGGCCGCCAACGT GCCAGATGCC TGGTGCACGC AGCAGAAGAT GAACTTCCGC GAGAAGGAGAAACAGGTGCT GGACCAGATC CTGGGGCCAG GTCGCTACGA CGCGCGCATC CGGCCGTCGGGGATCAACGG CACAGCGGAC AACCCCACCA TAGTGAAAGT GAACATCTTC CTTCGCTCTATCAGCAAAAT AGACGATTAT AAAATGATGT TCCGCTGCGC TGCCCCGCTT GCATGCCGCCCGGACCCCGC ACGCCGCCCT CGGCCCAGCG TCATGTGTTT TGTTACACGT GTTCTCAACAGGAGCCAGGC TCACCGACCC GGCCGCCAAT CAGGCGCCGC GTGTGCGACT CCGAACAAGGAATACAGCGT CCAGCTGACA TTCAGAGAGC AGTGGATGGA TGAGCGGCTC AAGTTCAATGACTTCAAAGG AAAAATAAAG TACCTGACAC TGACAGACGC CAACAGAGTA TGGATGCCAGATTTATTTTT CTCTAACGAA AAGGAAGGAC ATTTTCATAA CATTATCATG CCCAACGTTTATATACGTAT TTTTCCTTAT GGATCGGTGC TCTACAGTAT CAGAATCTCT CTGACSCTCTCCTGCCCGAT GAACCTGAAG CTGTACCCGC TCGACAGGCA GGTCTGCTCG CTGAGGATGGCCAGCTATGG TTGGACGACT GATGACCTGG TGTTCCTGTG GAAAGACGGC GACCCGGTGCAGGTGGTCAA GAACCTGCAC CTGCCGCGCT TCACGCTAGA GAAGTTCCTT ACCGACTACTGCAACAGCAA AACTAACACA GGAGAGTACA GCTGCTTGAA GGTGGACCTG CTGTTCAAGCGCGAGTTCAG CTACTACCTG ATCCAGATCT ACATCCCGTG CTGCATGCTG GTGATAGTGTCGTGGGTGTC CTTCTGGCTC GATCAGAGCG CCATCCCGGC ACGGGTGTCC CTAGGCGTGACCACGCTGCT CACCATGGCC ACCCAGACGT CGGGCATCAA CGCCTCCCTG CCCCCCGTGTCCTATACCAA AGCCATCGAC GTGTGGACTG GCGTGTGCCT CACTTTCGTG TTCGGCGCGTTGCTCGAGTT CGCGCTCGTC AACTACGCGA GCCGCTCGGA CATGCACCGC GAGAACATGAAGAAGCAGCG CCGCCAGGTA GAGCTGGAGC ACGCCGCGCA GCTCGAGGCG GCCGCTGACCTGCTCGTGGA GGACGGCAGC ACCACCTTTG CCATGAAGCC GTTGGTGGGA CACCCGGGCGGTCCGGCGGC CGCAGCGCCC GGCCTCGCAG GCCTGGACAA GGTGCGCCAG TGCGAGATCCACATGCAGCC CAAGCGCGAA AACTGCTGCC GCACCTGGCT CTCCAAGTTC CCCACGCGCTCCAAGCGCAT CGACGTCATC TCGCGCATCA CCTTCCCGCT CGTCTTCGCG CTCTTCAACCTCGTCTACTG GTCCACCTAC CTGTTCCGCG AGGACGACCG CGAGTGAGCC GCCAGCCGCCATCGCAGCAG CAGCCAGCT (SEQ ID NO:3).

The DNA molecule described in FIG. 1A–F as SAGluCl1SA (Sa1SA) and setforth as SEQ ID NO:5, which encodes the grasshopper GluCl proteindescribed in FIGS. 2A–B as 1SA and set forth as SEQ ID NO:6, thenucleotide sequence as follows:

AGCAGTGGCG CGACTACCGC CGCACGTGCC GCACCCCTCG CCACGCCGCA CCGCAGGTCTCTTCGCTCAT GGAGGAGCTC GTGCGACTGG AGTGAGCGTG TGGCTTACGG CTCTATCCGAGGAACCATAA GCAACAGTCA CCGGCAGTTG AGGGTTAACT GGTAATGCGG GCGCGGGTCGAGGCCAGCAG AGACCGCCGC GTGCCGCCCG CGGACTCGCA CTGCCCCCCG GACGCGCCGCCCCGGCCGCC AGCCGCGCGC TCGCCCTGCT CCCGCCGCCT CTGAAGTCCA AGGGTTAGCTCAGCCTCAGC ATCCCTGCAG GAACCATGAT GAGCGACCTC TGCAAGACGC CGTGGTGGCTTTGTGCGCTG CTGTTGCTGG CCGCCAACGT GCCAGATGCC TGGTGCACGC AGCAGAAGATGAACTTCCGC GAGAAGGAGA AACAGGTGCT GGACCAGATC CTGGGGCCAG GTCGCTACGACGCTCGCATC CGGCCGTCGG GGATCAACGG CACAGCGGAC AACCCCACCA TAGTGAAAGTGAACATCTTC CTTCGCTCTA TCAGCAAAAT AGACGATTAT AAAATGGAAT ACAGCGTCCAGCTGACATTC AGAGAGCAGT GGATGGATGA GCGGCTCAAG TTCAATGACT TCAAAGGAAAAATAAAGTAC CTGACACTGA CAGACGCCAA CAGAGTATGG ATGCCAGATT TATTTTTCTCTAACGAAAAG GAAGGACATT TTCATAACAT TATCATGCCC AACGTTTATA TACGTATTTTTCCTTATGGA TCGGTGCTCT ACAGTATCAG AATCTCTCTG ACGCTCTCCT GCCCGATGAACCTGAAGCTG TACCCGCTCG ACAGGCAGGT CTGCTCGCTG AGGATGGCCA GCTATGGTTGGACGACTGAT GACCTGGTGT TCCTGTGGAA AGACGGCGAC CCGGTGCAGG TGGTCAAGAACCTGCACCTG CCGCGCTTCA CGCTAGAGAA GTTCCTTACC GACTACTGCA ACAGCAAAACCAACACAGGA GAGTACAGCT GCTTGAAGGT GGACCTGCTG TTCAAGCGCG AGTTCAGCTACTACCTGATC CAGATCTACA TCCCGTGCTG CATGCTGGTG ATAGTGTCGT GGGTGTCCTTCTGGCTCGAT CAGAGCGCCA TCCCGGCACG GGTGTCCCTA GGCGTGACCA CGCTGCTCACCATGGCCACC CAGACGTCGG GCATCAACGC CTCCCTGCCC CCCGTGTCCT ATACCAAAGCCATCGACGTG TGGACTGGCG TGTGCCTCAC TTTCGTGTTC GGCGCGTTGC TCGAGTTCGCGCTCGTCAAC TACGCGAGCC GCTCGGACAT GCACCGCGAG AACATGAAGA AGCAGCGCCGCCAGGTAGAG CTGGAGCACG CCGCGCAGCT CGAGGCGGCC GCTGACCTGC TCGTGGAGGACGGCAGCACC ACCTTTGCCA TGAAGCCGTT GGTGGGACAC CCGGGCGGTC CGGCGGCCGCAGCGCCCGGC CTCGCAGGCC TGGACAAGGT GCGCCAGTGC GAGATCCACA TGCAGCCCAAGCGCGAAAAC TGCTGCCGCA CCTGGCTCTC CAAGTTCCCC ACGCGCTCCA AGCGCATCGACGTCATCTCG CGCATCACCT TCCCGCTCGT CTTCGCGCTC TTCAACCTCG TCTACTGGTCCACCTACCTG TTCCGCGAGG ACGAACGCGA GTGAGCCGCC AGCCGCCATC GCAGCAGCAGCCAAGCTACG TGGGGACGTC ATCGCC (SEQ ID NO:5).

The DNA molecule described in FIG. 1A–F as SAGluCl1SC (Sa1SC) and setforth as SEQ ID NO:7, which encodes the grasshopper GluCl proteindescribed in FIGS. 2A–B as 1SC and set forth as SEQ ID NO:8, thenucleotide sequence as follows:

AGCAGTGGCG CGACTACCGC CGCACGTGCC GCACGCCTCG CCACGCCGCA CCGCAGGTCTCTTCGCTCAT GGAGGAGCTC GTGCGACTGG AGTGAGCGTG TGGCTTACGG CTCTATCCGAGGAACCATAA GCAACAGTCA CCGGCAGTTG AGGGTTAACT GGTAATGCGG GCGCGGGTCGAGGCCAGCAG AGACCGCCGC GTGCCGCCCG CGGACTCGCA CTGCCCCCCG GACGCGCCGCCCCGGCCGCC AGCCGCGCGC TCGCCCTGCT CCCGCCGCCT CTGAAGTCCA AGGGTTAGCTCAGCCTCAGC ATCCCTGCAG GAACCATGAT GAGCGACCTC TGCAAGACGC CGTGGTGGCTTTGTGCGCTG CTGTTGCTGG CCGCCAACGT GCCAGATGCC TGGTGCACGC AGCAGAAGATGAACTTCCGC GAGAAGGAGA AACAGGTGCT GGACCAGATC CTGGGGCCAG GTCGCTACGACGCTCGCATC CGGCCGTCGG GGATCAACGG CACAGCGGAC AACCCCACCA TAGTGAAAGTGAACATCTTC CTTCGCTCTA TCAGCAAAAT AGACGATTAT AAAATGGAAT ACAGCGTCCAGCTGACATTC AGAGAGCAGT GGATGGATGA GCGGCTCAAG TTCAATGACT TCAAAGGAAAAATAAAGTAC CTGACACTGA CAGACGCCAA CAGAGTATGG ATGCCAGATT TATTTTTCTCTAACGAAAAG GAAGGACATT TTCATAACAT TATCATGCCC AACGTTTATA TACGTATTTTTCCTTATGGA TCGGTGCTCT ACAGTATCAG AATCTCTCTG ACGCTCTCCT GCCCGATGAACCTGAAGCTG TACCCGCTCG ACAGGCAGGT CTGCTCGCTG AGGATGGCCA GCTATGGTTGGACGACTGAT GACCTGGTGT TCCTGTGGAA AGACGGCGAC CCGGTGCAGG TGGTCAAGAACCTGCACCTG CCGCGCTTCA CGCTAGAGAA GTTCCTTACC GACTACTGCA ACAGCAAAACCAACACAGGA GAGTACAGCT GCTTGAAGGT GGACCTGCTG TTCAAGCGCG AGTTCAGCTACTACCTGATC CAGATCTACA TCCCGTGCTG CATGCTGGTG ATAGTGTCGT GGGTGTCCTTCTGGCTCGAT CAGAGCGCCA TCCCGGCACG GGTGTCCCTA GGCGTGACCA CGCTGCTCACCATGGCCACC CAGACGTCGG GCATCAACGC CTCCCTGCCC CCCGTGTCCT ATACCAAAGCCATCGACGTG TGGACTGGCG TGTGCCTCAC TTTCGTGTTC GGCGCGTTGC TCGAGTTCGCGCTCGTCAAC TACGCGAGCC GCTCGGACAT GCACCGCGAG AACATGAAGA AGCAGCGCCGCCAGGTAGAG CTGGAGCACG CCGCGCAGCT CGAGGCGGCC GCTGACCTGC TCGTGGAGGACGGCAGCACC ACCTTTGCCA TGAAGCCGTT GGTGGGACAC CCGGGCGGTC CGGCGGCCGCAGCGCCCGGC CTCGCAGGCC TGGACAAGGT GCGCCAGTGC GAGATCCACA TGCAGCCCAAGCGCGAAAAC TGCTGCCGCA CCTGGCTCTC CAAGTTCCCC ACGCGCTCCA AGCGCATCGACGTCATCTCG CGCATCACCT TCCCGCTCGT CTTCGCGCTC TTCAACCTCG TCTACTGGTCCACCTACCTG TTCCGCGAGG ACGACCGCGA GTGAGCCGCC AGCCGCCATC GCAGCAGCAGCCAAGCTACG TGGGGACGTC ATCGCC (SEQ ID NO:7).

The DNA molecule described in FIG. 1A–F as SaGluCl2 and set forth as SEQID NO:9, which encodes the grasshopper GluCl protein described in FIG.2A–B as “2” and set forth as SEQ ID NO:10, the nucleotide sequence asfollows:

GGCACGAGGG CGTCTCGGCT GTCCACACAC AGCAGGAATC ATGCTGAGCG CAACACCCAGCAAGCTGCGG CGATTTTGTG CTTTGGTGCT GTTGGTTGTG AATCTGTCAA AAGTCACATGCTCGGATCAG AAGACTAACA TCCGGGAGGC GGAGAGGCAG GTGATGGAGC ACGTCCTGAGCCCGAGCCCGC TACGACGCGC GGCTGCGGCC CCCGGGATAC AACGGCACAG AGAGTCCCACTGTGGTAAAA GTTAACATCT TTGTACGCTC CATCAGCAGA GTAGATGATC AGCATATGGAATATGACGCG CAGTTGACGT TCAGAGAGCA GTGGTTTGAC GACAGGCTCA AGTTTGACGATTTTGGAGGT AAAATCAAAT ATCTAACACT GACAGATCCC AGCAGAATAT GGATGCCAGATTTATTTTTT AGCAACGAGA AGAAAGCACA CTTTCACGAT GTAGTAATGC CTAATGTCTATGTACGAATA TTTCCTCTTG GGTCGGTTCT CTACAGTACC AGAATCTCAA TGACACTCTCGTGTCCCATG GACCTGAGGC TGTACCCACA CGATCGACAG GTGTGCTCCA TCAGGATGGCCAGCTATGGT TGGACGACTG AAGACCTGGT GTTCATGTGG AAAGACGGCG ACCCGGTGCAGGTGGTCAAG AACCTGCGCC TTCCACGCTT CACGCTAGAG AAGTTCGTTA CTGATTACTGCCACGCCAGG ACAAACACAG GCGAGTACAG CTGCCTTAAA GTGGAACTGG TGTTTAAACGCGAGTTCCGC TACTACATGG TGCACATCTA CATTCCGACT TTCATGCTGG TGATAGTGTCGTGGCTATCG TTCTGGCTGG ATCAGAGAGC CATCACGGCA CGGACGTGCC TGGTGGTCACGACGGTGCTC ACCATCACCA TCCAGACCTC GGGCGTCAGA AAGTCGCTGC CCGTCGTCAACTACGTCATG GCCGTCGAGG TGTGGCTCGG GATGTGCGTC TCGTTCGTGT TCGGCGCACTGCTGCAGCTG GCGCTGGTCA ACTACCTGGC CCGCAAAGAG GCGTGCCGAG GCGCCGCCAAGCGGCACAAC CGCCTCGTCG GCCCGGAGCA CTCTGCACAG CTGGAGGAGA TCAGCGAGGCTCTCGTCGAG GACGGCAGCG CGGCATTCGC TATGAAGCTG CTGGAGGAGC AGTCAAACAGCCCCACGGCT GACAAGGAGG CGGCGGCCCA GGGCGGAAGG TGCTCGCCGC GGCAGTGGTGGCGCTCCTGG ATGGCCAGCT TCCCCACGGG CTCGCAGCGC GCCGATGCCG CATCGCGCGTCCTCTTCCCT CTCGCCTTCT CCCTCTTCTG CCTCGCCTAC TGGTGTGTCA ACGCCTCCGGATAACAGCAA CACCAGTTGC TCGGCAGTGT CCTACGATGG TGGTGGAAGT AACCCTCCACACATATTTGC AGATGCACAA GCATTTATAC CTATTATAGA ATGAAAAATA AGTATTAAAGTACTGTATAA TTCTCTATTC ATCATTACTC TTACTGTATC GACATTACTG TTTGACGGCAATTTCGTTAT ACGAGATTTG TCCGGAAAAT ACGCATAAAA GTTGAATTAT AACTTTATTTTACAATTATT CAGGTATTAC AATATGGTCT CCTTCAAAGT ACTCGCCCTG AGATGCGATACACTGCTGTC AACGCCGTTT ACACTGTTGA TAATATTGCT GAAAGTCTTC AGTTGTGATGTTGTTCAGTT GCCGTGTCGT TTCCGCCTTG TGGCTTCCAC ATCTCCCAAG TGCCGCCCTCGAAGCACGAT TTTGCATTTC TGGAACATGA AGAAGTCACA TGGGGCTAAA GCGGGTGAATAAGGCGGGTG GTCTGTCACG GTGATTGAGC TTCAGGCCAA AAACTCGCGC ACAACGAGCGACGTGTGAGC GGGCGCTTTG TCGTGCTGAA GGATCCACCT GCCCCCTTTT GCCAACTCCGGTCGAACTCG GGCCACACGA GCTCTGAGAC GTGTCAGAAC TTCAACGTAA AAATTTCCGTTCACTCTCTG GCCGGGAAGG ACAAATTCCT TGTG (SEQ ID NO:9).

The above-exemplified isolated DNA molecules, shown in FIG. 1A–Fcomprise the following characteristics:

SAGluCl1L (Sa1LA; SEQ ID NO:1):

1879 nuc.: initiating Met (nuc. 306–308) and “TGA” term. codon (nuc.1845–1847);

SAGluCl1L (Sa1LC; SEQ ID NO:3):

1879 nuc.: initiating Met (nuc. 306–308) and “TGA” term. codon (nuc.1845–1847);

SAGluCl1S (Sa1SA; SEQ ID NO:5):

1776 nuc.: initiating Met (nuc. 326–328) and “TGA” term. codon (nuc.1712–1713);

SAGluCl1S (Sa1SC; SEQ ID NO:7):

1776 nuc.: initiating Met (nuc. 326–328) and “TGA” term. codon (nuc.1712–1713);

SAGluCl2 (Sa2; SEQ ID NO:9):

2074 nuc.: initiating Met (nuc. 41–43) and “TGA” term. codon (nuc.1382–1384).

In regard to the “long” form of SaGluCl1, the “LA”cDNA (SEQ ID NO:1)encodes a protein with a Glu residue at amino acid number 511, whereasthe “LC cDNA (SEQ ID NO:3) encodes a protein with a Asp residue at aminoacid number 511.

In regard to the “short” form of SaGluCl1, the “SA”cDNA (SEQ ID NO:5)encodes a protein with a Glu residue at amino acid number 460, whereasthe “SC cDNA (SEQ ID NO:7) encodes a protein with a Asp residue at aminoacid number 460.

The present invention also relates to biologically active fragments ormutants of SEQ ID NOs:1, 3, 5, 7 and 9 which encode mRNA expressingSaGluCl1 or SaGluCl2, respectively. Any such biologically activefragment and/or mutant will encode either a protein or protein fragmentwhich at least substantially mimics the wild type protein, including butnot limited to the wild type forms as set forth in SEQ ID NOs:2, 4, 6, 8and/or 10. Any such polynucleotide includes but is not necessarilylimited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a protein or protein fragmentof diagnostic, therapeutic or prophylactic use and would be useful forscreening for agonists and/or antagonists for SaGluCl function.

A preferred aspect of this portion of the present invention is disclosedin FIGS. 1A–F, which describes the cDNA molecule encoding various formsof SaGluCl channel proteins.

The isolated nucleic acid molecules of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

The degeneracy of the genetic code is such that, for all but two aminoacids, more than a single codon encodes a particular amino acid. Thisallows for the construction of synthetic DNA that encodes the SaGluClprotein where the nucleotide sequence of the synthetic DNA differssignificantly from the nucleotide sequence of SEQ ID NOs:1, 3, 5, 7 and9 but still encodes the same SaGluCl protein as SEQ ID NO:1, 3, 5, 7,and 9. Such synthetic DNAs are intended to be within the scope of thepresent invention. If it is desired to express such synthetic DNAs in aparticular host cell or organism, the codon usage of such synthetic DNAscan be adjusted to reflect the codon usage of that particular host, thusleading to higher levels of expression of the SaGluCl channel protein inthe host. In other words, this redundancy in the various codons whichcode for specific amino acids is within the scope of the presentinvention. Therefore, this invention is also directed to those DNAsequences which encode RNA comprising alternative codons which code forthe eventual translation of the identical amino acid, as shown below:

-   A=Ala=Alanine: codons GCA, GCC, GCG, GCU-   C=Cys=Cysteine: codons UGC, UGU-   D=Asp=Aspartic acid: codons GAC, GAU-   E=Glu=Glutamic acid: codons GAA, GAG-   F=Phe=Phenylalanine: codons UUC, UUU-   G=Gly=Glycine: codons GGA, GGC, GGG, GGU-   H=His=Histidine: codons CAC, CAU-   I=Ile=Isoleucine: codons AUA, AUC, AUU-   K=Lys=Lysine: codons AAA, AAG-   L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU-   M=Met=Methionine: codon AUG-   N=Asp=Asparagine: codons AAC, AAU-   P=Pro=Proline: codons CCA, CCC, CCG, CCU-   Q=Gln=Glutamine: codons CAA, CAG-   R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU-   S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU-   T=Thr=Threonine: codons ACA, ACC, ACG, ACU-   V=Val=Valine: codons GUA, GUC, GUG, GUU-   W=Trp=Tryptophan: codon UGG-   Y=Tyr=Tyrosine: codons UAC, UAU    Therefore, the present invention discloses codon redundancy which    may result in differing DNA molecules expressing an identical    protein. For purposes of this specification, a sequence bearing one    or more replaced codons will be defined as a degenerate variation.    Also included within the scope of this invention are mutations    either in the DNA sequence or the translated protein which do not    substantially alter the ultimate physical properties of the    expressed protein. For example, substitution of valine for leucine,    arginine for lysine, or asparagine for glutamine may not cause a    change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate or a receptor for a ligand.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.,:(Computational Molecular Biology, Lesk, A. M., ed. Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds. HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exists a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo and Lipton, 1988, SIAM J Applied Math48:1073). Methods commonly employed to determine identity or similaritybetween two sequences include, but are not limited to, those disclosedin Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, SanDiego, 1994, and Carillo and Lipton, 1988, SIAM J Applied Math 48:1073.Methods to determine identity and similarity are codified in computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, GCGprogram package (Devereux, et al, 1984, Nucleic Acids Research12(1):387), BLASTN, and FASTA (Altschul, et al., 1990, J. Mol. Biol.215:403). As an illustration, by a polynucleotide having a nucleotidesequence having at least, for example, 95% “identity” to a referencenucleotide sequence of SEQ ID NO:1, 3, 5, 7 and/or 9 is intended thatthe nucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence may includeup to five point mutations or alternative nucleotides per each 100nucleotides of the reference nucleotide sequence of SEQ ID NO:1, 3, 5, 7and/or 9. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These mutations or alternative nucleotidesubstitutions of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. One source of such a “mutation” orchange which results in a less than 100% identity may occur through RNAediting. The process of RNA editing results in modification of an mRNAmolecule such that use of that modified mRNA as a template to generate acloned cDNA may result in one or more nucleotide changes, which may ormay not result in a codon change. This RNA editing is known to becatalyzed by an RNA editase. Such an RNA editase is RNA adenosinedeaminase, which converts an adenosine residue to an inosine residue,which tends to mimic a cytosine residue. To this end, conversion of anmRNA residue from A to I will result in A to G transitions in the codingand noncoding regions of a cloned cDNA (e.g., see Hanrahan et al, 1999,Annals New York Acad. Sci. 868:51–66; for a review see Bass, 1997, TIBS22: 157–162). Similarly, by a polypeptide having an amino acid sequencehaving at least, for example, 95% identity to a reference amino acidsequence of SEQ ID NO:2, 4, 6, 8 and/or 10 is intended that the aminoacid sequence of the polypeptide is identical to the reference sequenceexcept that the polypeptide sequence may include up to five amino acidalterations per each 100 amino acids of the reference amino acid of SEQID NO:2, 4, 6, 8 and/or 10. In other words, to obtain a polypeptidehaving an amino acid sequence at least 95% identical to a referenceamino acid sequence, up to 5% of the amino acid residues in thereference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceof anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. Again, as noted above,RNA editing may result in a codon change which will result in anexpressed protein which differs in “identity” from other proteinsexpressed from “non-RNA edited” transcripts, which correspond directlyto the open reading frame of the genomic sequence.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification. The nucleic acid molecules of the present inventionencoding a SaGluCl channel protein, in whole or in part, can be linkedwith other DNA molecules, i.e, DNA molecules to which the SaGluCl codingsequence are not naturally linked, to form “recombinant DNA molecules”which encode a respective SaGluCl channel protein. The novel DNAsequences of the present invention can be inserted into vectors whichcomprise nucleic acids encoding SaGluCl or a functional equivalent.These vectors may be comprised of DNA or RNA; for most cloning purposesDNA vectors are preferred. Typical vectors include plasmids, modifiedviruses, bacteriophage, cosmids, yeast artificial chromosomes, and otherforms of episomal or integrated DNA that can encode a SaGluCl channelprotein. It is well within the purview of the skilled artisan todetermine an appropriate vector for a particular gene transfer or otheruse.

Included in the present invention are DNA sequences that hybridize toSEQ ID NOs:1, 3, 5, 7 and 9 under stringent conditions. By way ofexample, and not limitation, a procedure using conditions of highstringency is as follows: Prehybridization of filters containing DNA iscarried out for 2 hours to overnight at 65° C. in buffer composed of6×SSC, 5× Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA.Filters are hybridized for 12 to 48 hrs at 65° C. in prehybridizationmixture containing 100 μg/ml denatured salmon sperm DNA and 5−20×10⁶ cpmof ³²P-labeled probe. Washing of filters is done at 37° C. for 1 hr in asolution containing 2×SSC, 0.1% SDS. This is followed by a wash in0.1×SSC, 0.1% SDS at 50° C. for 45 min. before autoradiography. Otherprocedures using conditions of high stringency would include either ahybridization step carried out in 5×SSC, 5× Denhardt's solution, 50%formamide at 42° C. for 12 to 48 hours or a washing step carried out in0.2× SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes.

Reagents mentioned in the foregoing procedures for carrying out highstringency hybridization are well known in the art. Details of thecomposition of these reagents can be found in, e.g., Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. In addition to the foregoing, otherconditions of high stringency which may be used are well known in theart.

The present invention also relates to a substantially purified form of arespective SaGluCl channel protein, which comprises the amino acidsequence disclosed in FIGS. 2A–B and as set forth in SEQ ID NOs:2, 4, 6,8 and 10. The disclosed SaGluCl proteins contain an open reading frameof 513 (SEQ ID NOs: 2 and 4), 462 amino acids (SEQ ID NOs: 6 and 8) and447 (SEQ ID NO:10) amino acids in length, as shown in FIGS. 2A–B, and asfollows:

MMSDLCKTPW WLCALLLLAA NVPDAWCTQQ KMNFREKEKQ VLDQILGPGR YDARIRPSGINGTADNPTIV KVNIFLRSIS KIDDYKMMFR CAAPLACRPD PARRPRPSVM CFVTRVLNRSQAHRPGRQSG AACATPNKEY SVQLTFREQW MDERLKFNDF KGKIKYLTLT DANRVWMPDLFFSNEKEGHF HNIIMPNVYI RIFPYGSVLY SIRISLTLSC PMNLKLYPLD RQVCSLRMASYGWTTDDLVF LWKDGDPVQV VKNLHLPRFT LEKFLTDYCN SKTNTGEYSC LKVDLLFKREFSYYLIQIYI PCCMLVIVSW VSFWLDQSAI PARVSLGVTT LLTMATQTSG INASLPPVSYTKAIDVWTGV CLTFVFGALL EFALVNYASR SDMHRENMKK QRRQVELEHA AQLEAAADLLVEDGSTTFAM KPLVGHPGGP AAAAPGLAGL DKVRQCEIHM QPKRENCCRT WLSKFPTRSKRIDVISRITF PLVFALFNLV YWSTYLFRED ERE (SEQ ID NO:2);MMSDLCKTPW WLCALLLLAA NVPDAWCTQQ KMNFREKEKQ VLDQILGPGR YDARIRPSGINGTADNPTIV KVNIFLRSIS KIDDYKMMFR CAAPLACRPD PARRPRPSVM CFVTRVLNRSQAHRPGRQSG AACATPNKEY SVQLTFREQW MDERLKFNDF KGKIKYLTLT DANRVWMPDLFFSNEKEGHF HNIIMPNVYI RIFPYGSVLY SIRISLTLSC PMNLKLYPLD RQVCSLRMASYGWTTDDLVF LWKDGDPVQV VKNLHLPRFT LEKFLTDYCN SKTNTGEYSC LKVDLLFKREFSYYLIQIYI PCCMLVIVSW VSFWLDQSAI PARVSLGVTT LLTMATQTSG INASLPPVSYTKAIDVWTGV CLTFVFGALL EFALVNYASR SDMHRENMKK QRRQVELEHA AQLEAAADLLVEDGSTTFAM KPLVGHPGGP AAAAPGLAGL DKVRQCEIHM QPKRENCCRT WLSKFPTRSKRIDVISRITF PLVFALFNLV YWSTYLFRED DRE (SEQ ID NO:4);MMSDLCKTPW WLCALLLLAA NVPDAWCTQQ KMNFREKEKQ VLDQILGPGR YDARIRPSGINGTADNPTIV KVNIFLRSIS KIDDYKMEYS VQLTFREQWM DERLKFNDFK GKIKYLTLTDANRVWMPDLF FSNEKEGHFH NIIMPNVYIR IFPYGSVLYS IRISLTLSCP MNLKLYPLDRQVCSLRMASY GWTTDDLVFL WKDGDPVQVV KNLHLPRFTL EKFLTDYCNS KTNTGEYSCLKVDLLFKREF SYYLIQIYIP CCMLVIVSWV SFWLDQSAIP ARVSLGVTTL LTMATQTSGINASLPPVSYT KAIDVWTGVC LTFVFGALLE FALVNYASRS DMHRENMKKQ RRQVELEHAAQLEAAADLLV EDGSTTFAMK PLVGHPGGPA AAAPGLAGLD KVRQCEIHMQ PKRENCCRTWLSKFPTRSKR IDVISRITFP LVFALFNLVY WSTYLFREDE RE (SEQ ID NO:6);MMSDLCKTPW WLCALLLLAA NVPDAWCTQQ KMNFREKEKQ VLDQILGPGR YDARIRPSGINGTADNPTIV KVNIFLRSIS KIDDYKMEYS VQLTFREQWM DERLKFNDFK GKIKYLTLTDANRVWMPDLF FSNEKEGHFH NIIMPNVYIR IFPYGSVLYS IRISLTLSCP MNLKLYPLDRQVCSLRMASY GWTTDDLVFL WKDGDPVQVV KNLHLPRFTL EKFLTDYCNS KTNTGEYSCLKVDLLFKREF SYYLIQIYIP CCMLVIVSWV SFWLDQSAIP ARVSLGVTTL LTMATQTSGINASLPPVSYT KAIDVWTGVC LTFVFGALLE FALVNYASRS DMHRENMKKQ RRQVELEHAAQLEAAADLLV EDGSTTFAMK PLVGHPGGPA AAAPGLAGLD KVRQCEIHMQ PKRENCCRTWLSKFPTRSKR IDVISRITFP LVFALFNLVY WSTYLFREDD RE (SEQ ID NO:8); and,MLSATPSKLR RFCALVLLVV NLSKVTCSDQ KTNIREAERQ VMEHVLSPSR YDARLRPPGYNGTESPTVVK VNIFVRSISR VDDQHMEYDA QLTFREQWFD DRLKFDDFGG KIKYLTLTDPSRIWMPDLFF SNEKKAHFHD VVMPNVYVRI FPLGSVLYST RISMTLSCPM DLRLYPHDRQVCSIRMASYG WTTEDLVFMW KDGDPVQVVK NLRLPRFTLE KFVTDYCHAR TNTGEYSCLKVELVFKREFR YYMVHIYIPT FMLVIVSWLS FWLDQRAITA RTCLVVTTVL TITIQTSGVRKSLPVVNYVM AVEVWLGMCV SFVFGALLQL ALVNYLARKE ACRGAAKRHN RLVGPEHSAQLEEISEALVE DGSAAFAMKL LEEQSNSPTA DKEAAAQGGR CSPRQWWRSW MASFPTGSQRADAASRVLFP LAFSLFCLAY WCVNASG (SEQ ID NO:10).

The present invention also relates to biologically active fragmentsand/or mutants of the SaGluCl protein comprising the amino acid sequenceas set forth in SEQ ID NOs:2, 4, 6, 8, and 10, including but notnecessarily limited to amino acid substitutions, deletions, additions,amino terminal truncations and carboxy-terminal truncations such thatthese mutations provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use and would be useful for screening foragonists and/or antagonists of SaGluCl function.

Another preferred aspect of the present invention relates to asubstantially purified, fully processed GluCl channel protein obtainedfrom a recombinant host cell containing a DNA expression vectorcomprises a nucleotide sequence as set forth in SEQ ID NOs: 1, 3, 5, 7,and/or 9 and expresses the respective SaGluCl precursor protein. It isespecially preferred is that the recombinant host cell be a eukaryotichost cell, such as a mammalian cell line, or Xenopus oocytes, as notedabove.

As with many proteins, it is possible to modify many of the amino acidsof SaGluCl channel protein and still retain substantially the samebiological activity as the wild type protein. Thus this inventionincludes modified SaGluCl polypeptides which have amino acid deletions,additions, or substitutions but that still retain substantially the samebiological activity as a respective, corresponding SaGluCl. It isgenerally accepted that single amino acid substitutions do not usuallyalter the biological activity of a protein (see, e.g., Molecular Biologyof the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/CummingsPublishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science244:1081–1085). Accordingly, the present invention includes polypeptideswhere one amino acid substitution has been made in SEQ ID NO:2, 4, 6, 8and/or 10 wherein the polypeptides still retain substantially the samebiological activity as a corresponding SaGluCl protein. The presentinvention also includes polypeptides where two or more amino acidsubstitutions have been made in SEQ ID NO:2, 4, 6, 8 or 10 wherein thepolypeptides still retain substantially the same biological activity asa corresponding SaGluCl protein. In particular, the present inventionincludes embodiments where the above-described substitutions areconservative substitutions.

One skilled in the art would also recognize that polypeptides that arefunctional equivalents of SaGluCl and have changes from the SaGluClamino acid sequence that are small deletions or insertions of aminoacids could also be produced by following the same guidelines, (i.e,minimizing the differences in amino acid sequence between SaGluCl andrelated proteins. Small deletions or insertions are generally in therange of about 1 to 5 amino acids. The effect of such small deletions orinsertions on the biological activity of the modified SaGluClpolypeptide can easily be assayed by producing the polypeptidesynthetically or by making the required changes in DNA encoding SaGluCland then expressing the DNA recombinantly and assaying the proteinproduced by such recombinant expression.

The present invention also includes truncated forms of SaGluCl whichcontain the region comprising the active site of the enzyme. Suchtruncated proteins are useful in various assays described herein, forcrystallization studies, and for structure-activity-relationshipstudies.

The present invention also relates to crude or substantially purifiedsubcellular membrane fractions from the recombinant host cells (bothprokaryotic and eukaryotic as well as both stably and transientlytransformed cells) which contain the nucleic acid molecules of thepresent invention. These recombinant host cells express SaGluCl or afunctional equivalent, which becomes post translationally associatedwith the cell membrane in a biologically active fashion. Thesesubcellular membrane fractions will comprise either wild-type or mutantforms of SaGluCl at levels substantially above endogenous levels andhence will be useful in assays to select modulators of SaGluCl proteinsor channels. In other words, a specific use for such subcellularmembranes involves expression of SaGluCl within the recombinant cellfollowed by isolation and substantial purification of the membranes awayfrom other cellular components and subsequent use in assays to selectfor modulators, such as agonist or antagonists of the protein orbiologically active channel comprising one or more of the proteinsdisclosed herein. Therefore, another preferred aspect of the presentinvention relates to a substantially purified membrane preparation whichhas been obtained from a recombinant host cell transformed ortransfected with a DNA expression vector which comprises andappropriately expresses a complete open reading frame as set forth inSEQ ID NOs: 1, 3, 5, 7, and/or 9, resulting in a functional, processedform of the respective SaGluCl channel. It is especially preferred isthat the recombinant host cell be a eukaryotic host cell, such as amammalian cell line, or Xenopus oocytes, as noted above.

The present invention also relates to isolated nucleic acid moleculeswhich are fusion constructions expressing fusion proteins useful inassays to identify compounds which modulate wild-type SaGluCl activity,as well as generating antibodies against SaGluCl. One aspect of thisportion of the invention includes, but is not limited to, glutathioneS-transferase (GST)-SaGluCl fusion constructs. Recombinant GST-SaGluClfusion proteins may be expressed in various expression systems,including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using abaculovirus expression vector (pAcG2T, Pharmingen). Another aspectinvolves SaGluCl1 and/or SaGlu-2 fusion constructs linked to variousmarkers, including but not limited to GFP (Green fluorescent protein),the MYC epitope, and GST. Again, any such fusion constructs may beexpressed in the cell line of interest and used to screen for modulatorsof one or more of the SaGluCl proteins disclosed herein.

A preferred aspect for screening for modulators of SaGluCl channelactivity is an expression system for the electrophysiological-basedassays for measuring glutamate-gated chloride channel activitycomprising injecting the DNA molecules of the present invention intoXenopus laevis oocytes. The general use of Xenopus oocytes in the studyof ion channel activity is known in the art (Dascal, 1987, Crit. Rev.Biochem. 22: 317–317; Lester, 1988, Science 241: 1057–1063; see alsoMethods of Enzymology, Vol. 207, 1992, Ch. 14–25, Rudy and Iverson, ed.,Academic Press, Inc., New York). A portion of the present inventiondiscloses an improved method of measuring channel activity andmodulation by agonists and/or antagonists which is several-fold moresensitive than previously disclosed. The Xenopus oocytes are injectedwith nucleic acid material, including but not limited to DNA, mRNA orcRNA which encode a gated-channel, wherein channel activity may bemeasured as well as response of the channel to various modulators. Ionchannel activity is measured by utilizing a holding potential morepositive than the reversal potential for chloride (i.e, greater than −30mV), preferably about 0 mV. This alteration in assay measurementconditions has resulting in a 10-fold increase in sensitivity of theassay to modulation by ivermectin phosphate. Therefore, this improvedassay allows screening and selecting for compounds which modulate GluClactivity at levels which were previously thought to be undetectable.This procedure is outlined in the Example section. It will be evident tothe skilled artisan that this method may be utilized in various ionchannel measurement assays, and especially assays which measureglutamate-gated activity in a eukaryotic cell, such as a Xenopus oocyte.It is especially preferred that invertebrate glutamate-gated chloridechannels, including but in no way limited to Caenorhabditis elegans,Drosophila melonogaster, Ctenocephalides felis glutamate-gated channelsas well as the S. americana glutamate-gated channel proteins disclosedherein, may be utilized in an assay to screen and select for compoundswhich modulate the activity of these channels. Levels of SaGluCl proteinin host cells are quantified by immunoaffinity and/or ligand affinitytechniques. Cells expressing SaGluCl can be assayed for the number ofGluCl molecules expressed by measuring the amount of radioactiveglutamate or ivermectin binding to cell membranes. SaGluCl-specificaffinity beads or SaGluCl-specific antibodies are used to isolate forexample ³⁵S-methionine labeled or unlabelled SaGluCl protein. LabeledSaGluCl protein is analyzed by SDS-PAGE. Unlabelled SaGluCl protein isdetected by Western blotting, ELISA or RIA assays employing SaGluClspecific antibodies.

Any of a variety of procedures may be used to clone SaGluCl. Thesemethods include, but are not limited to, (1) a RACE PCR cloningtechnique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998–9002). 5′ and/or 3′ RACE may be performed to generate a full-lengthcDNA sequence. This strategy involves using gene-specificoligonucleotide primers for PCR amplification of SaGluCl cDNA. Thesegene-specific primers are designed through identification of anexpressed sequence tag (EST) nucleotide sequence which has beenidentified by searching any number of publicly available nucleic acidand protein databases; (2) direct functional expression of the SaGluClcDNA following the construction of a SaGluCl-containing cDNA library inan appropriate expression vector system; (3) screening aSaGluCl-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a labeled degenerate oligonucleotide probedesigned from the amino acid sequence of the SaGluCl protein; (4)screening a SaGluCl-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA encoding theSaGluCl protein. This partial cDNA is obtained by the specific PCRamplification of SaGluCl DNA fragments through the design of degenerateoligonucleotide primers from the amino acid sequence known for otherkinases which are related to the SaGluCl protein; (5) screening aSaGluCl-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a partial cDNA or oligonucleotide withhomology to a mammalian SaGluCl protein. This strategy may also involveusing gene-specific oligonucleotide primers for PCR amplification ofSaGluCl cDNA identified as an EST as described above; or (6) designing5′ and 3′ gene specific oligonucleotides using SEQ ID NO: 1, 3, 5, 7, or9 as a template so that either the full-length cDNA may be generated byknown RACE techniques, or a portion of the coding region may begenerated by these same known RACE techniques to generate and isolate aportion of the coding region to use as a probe to screen one of numeroustypes of cDNA and/or genomic libraries in order to isolate a full-lengthversion of the nucleotide sequence encoding SaGluCl.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cell types-orspecies types, may be useful for isolating a SaGluCl-encoding DNA or aSaGluCl homologue. Other types of libraries include, but are not limitedto, cDNA libraries derived from other cells.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have SaGluClactivity. The selection of cells or cell lines for use in preparing acDNA library to isolate a cDNA encoding SaGluCl may be done by firstmeasuring cell-associated SaGluCl activity using any known assayavailable for such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

It is also readily apparent to those skilled in the art that DNAencoding SaGluCl may also be isolated from a suitable genomic DNAlibrary. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in Sambrook, et al., supra.One may prepare genomic libraries, especially in P1 artificialchromosome vectors, from which genomic clones containing the SaGluCl canbe isolated, using probes based upon the SaGluCl nucleotide sequencesdisclosed herein. Methods of preparing such libraries are known in theart (Ioannou et al., 1994, Nature Genet. 6:84–89).

In order to clone a SaGluCl gene by one of the preferred methods, theamino acid sequence or DNA sequence of a SaGluCl or a homologous proteinmay be necessary. To accomplish this, a respective SaGluCl channelprotein may be purified and the partial amino acid sequence determinedby automated sequenators. It is not necessary to determine the entireamino acid sequence, but the linear sequence of two regions of 6 to 8amino acids can be determined for the PCR amplification of a partialSaGluCl DNA fragment. Once suitable amino acid sequences have beenidentified, the DNA sequences capable of encoding them are synthesized.Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the SaGluCl sequence butothers in the set will be capable of hybridizing to SaGluCl DNA even inthe presence of DNA oligonucleotides with mismatches. The mismatched DNAoligonucleotides may still sufficiently hybridize to the SaGluCl DNA topermit identification and isolation of SaGluCl encoding DNA.Alternatively, the nucleotide sequence of a region of an expressedsequence may be identified by searching one or more available genomicdatabases. Gene-specific primers may be used to perform PCRamplification of a cDNA of interest from either a cDNA library or apopulation of cDNAs. As noted above, the appropriate nucleotide sequencefor use in a PCR-based method may be obtained from SEQ ID NO: 1, 3, 5, 7or 9 either for the purpose of isolating overlapping 5′ and 3′ RACEproducts for generation of a full-length sequence coding for SaGluCl, orto isolate a portion of the nucleotide sequence coding for SaGluCl foruse as a probe to screen one or more cDNA- or genomic-based libraries toisolate a full-length sequence encoding SaGluCl or SaGluCl-likeproteins.

This invention also includes vectors containing a SaGluCl gene, hostcells containing the vectors, and methods of making substantially pureSaGluCl protein comprising the steps of introducing the SaGluCl geneinto a host cell, and cultivating the host cell under appropriateconditions such that SaGluCl is produced. The SaGluCl so produced may beharvested from the host cells in conventional ways. Therefore, thepresent invention also relates to methods of expressing the SaGluClprotein and biological equivalents disclosed herein, assays employingthese gene products, recombinant host cells which comprise DNAconstructs which express these proteins, and compounds identifiedthrough these assays which act as agonists or antagonists of SaGluClactivity.

The cloned SaGluCl cDNA obtained through the methods described above maybe recombinantly expressed by molecular cloning into an expressionvector (such as pcDNA3.neo, pcDNA3.1, pCR2.1, pBlueBacHis2 or pLITMUS28)containing a suitable promoter and other appropriate transcriptionregulatory elements, and transferred into prokaryotic or eukaryotic hostcells to produce recombinant SaGluCl. Expression vectors are definedherein as DNA sequences that are required for the transcription ofcloned DNA and the translation of their mRNAs in an appropriate host.Such vectors can be used to express eukaryotic DNA in a variety of hostssuch as bacteria, blue green algae, plant cells, insect cells and animalcells. Specifically designed vectors allow the shuttling of DNA betweenhosts such as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one which causes mRNAs to be initiated at highfrequency. To determine the SaGluCl cDNA sequence(s) that yields optimallevels of SaGluCl, cDNA molecules including but not limited to thefollowing can be constructed: a cDNA fragment containing the full-lengthopen reading frame for SaGluCl as well as various constructs containingportions of the cDNA encoding only specific domains of the protein orrearranged domains of the protein. All constructs can be designed tocontain none, all or portions of the 5′ and/or 3′ untranslated region ofa SaGluCl cDNA. The expression levels and activity of SaGluCl can bedetermined following the introduction, both singly and in combination,of these constructs into appropriate host cells. Following determinationof the SaGluCl cDNA cassette yielding optimal expression in transientassays, this SaGluCl cDNA construct is transferred to a variety ofexpression vectors (including recombinant viruses), including but notlimited to those for mammalian cells, plant cells, insect cells,oocytes, bacteria, and yeast cells. Techniques for such manipulationscan be found described in Sambrook, et al., supra, are well known andavailable to the artisan of ordinary skill in the art. Therefore,another aspect of the present invention includes host cells that havebeen engineered to contain and/or express DNA sequences encoding theSaGluCl. An expression vector containing DNA encoding a SaGluCl-likeprotein may be used for expression of SaGluCl in a recombinant hostcell. Such recombinant host cells can be cultured under suitableconditions to produce SaGluCl or a biologically equivalent form.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.Commercially available mammalian expression vectors which may besuitable for recombinant SaGluCl expression, include but are not limitedto, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega),pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs),pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMC1neo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),pUCTag (ATCC 37460), and IZD35 (ATCC 37565). Also, a variety ofbacterial expression vectors may be used to express recombinant SaGluClin bacterial cells. Commercially available bacterial expression vectorswhich may be suitable for recombinant SaGluCl expression include, butare not limited to pCR2.1 (Invitrogen), pET11a (Novagen), lambda gt11(Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungalcell expression vectors may be used to express recombinant SaGluCl infungal cells. Commercially available fungal cell expression vectorswhich may be suitable for recombinant SaGluCl expression include but arenot limited to pYES2 (Invitrogen) and Pichia expression vector(Invitrogen). Also, a variety of insect cell expression vectors may beused to express recombinant protein in insect cells. Commerciallyavailable insect cell expression vectors which may be suitable forrecombinant expression of SaGluCl include but are not limited topBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to, bacteria such as E. coli, fungal cells such as yeast,mammalian cells including, but not limited to, cell lines of bovine,porcine, monkey and rodent origin; and insect cells including but notlimited to Drosophila and silkworm derived cell lines. For instance, oneinsect expression system utilizes Spodoptera frugiperda (Sf21) insectcells (Invitrogen) in tandem with a baculovirus expression vector(pAcG2T, Pharmingen). Also, mammalian species which may be suitable andwhich are commercially available, include but are not limited to, Lcells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCCHTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCCCRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL209).

As noted above in regard to the use of Xenopus oocytes to express aSaGluCl gene of interest, the present invention is directed to methodsfor screening for compounds which modulate the expression of DNA or RNAencoding a SaGluCl protein. Compounds which modulate these activitiesmay be DNA, RNA, peptides, proteins, or non-proteinaceous organicmolecules. Compounds may modulate by increasing or attenuating theexpression of DNA or RNA encoding SaGluCl, or the function of theSaGluCl-based channels. Compounds that modulate the expression of DNA orRNA encoding SaGluCl or the biological function thereof may be detectedby a variety of assays. The assay may be a simple “yes/no” assay todetermine whether there is a change in expression or function. The assaymay be made quantitative by comparing the expression or function of atest sample with the levels of expression or function in a standardsample. Kits containing SaGluCl, antibodies to SaGluCl, or modifiedSaGluCl may be prepared by known methods for such uses.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels ofSaGluCl. The recombinant proteins, DNA molecules, RNA molecules andantibodies lend themselves to the formulation of kits suitable for thedetection and typing of SaGluCl. Such a kit would comprise acompartmentalized carrier suitable to hold in close confinement at leastone container. The carrier would further comprise reagents such asrecombinant SaGluCl or anti-SaGluCl antibodies suitable for detectingSaGluCl. The carrier may also contain a means for detection such aslabeled antigen or enzyme substrates or the like.

The assays described herein can be carried out with cells that have beentransiently or stably transfected with SaGluCl. The expression vectormay be introduced into host cells via any one of a number of techniquesincluding but not limited to transformation, transfection, protoplastfusion, and electroporation. Transfection is meant to include any methodknown in the art for introducing SaGluCl into the test cells. Forexample, transfection includes calcium phosphate or calcium chloridemediated transfection, lipofection, infection with a retroviralconstruct containing SaGluCl, and electroporation. The expressionvector-containing cells are individually analyzed to determine whetherthey produce SaGluCl protein. Identification of SaGluCl expressing cellsmay be done by several means, including but not limited to immunologicalreactivity with anti-SaGluCl antibodies, labeled ligand binding, thepresence of host cell-associated SaGluCl activity via ligand binding.

The specificity of binding of compounds showing affinity for SaGluCl isshown by measuring the affinity of the compounds for recombinant cellsexpressing the cloned receptor or for membranes from these cells.Expression of the cloned receptor and screening for compounds that bindto SaGluCl or that inhibit the binding of a known, radiolabeled ligandof SaGluCl to these cells, or membranes prepared from these cells,provides an effective method for the rapid selection of compounds withhigh affinity for SaGluCl. Such ligands need not necessarily beradiolabeled but can also be nonisotopic compounds that can be used todisplace bound radiolabeled compounds or that can be used as activatorsin functional assays. Compounds identified by the above method arelikely to be agonists or antagonists of SaGluCl and may be peptides,proteins, or non-proteinaceous organic molecules.

Accordingly, the present invention is directed to methods for screeningfor compounds which modulate the expression of DNA or RNA encoding aSaGluCl protein as well as compounds which effect the function of theSaGluCl protein. Methods for identifying agonists and antagonists ofother receptors are well known in the art and can be adapted to identifyagonists and antagonists of SaGluCl. For example, Cascieri et al. (1992,Molec. Pharmacol. 41:1096–1099) describe a method for identifyingsubstances that inhibit agonist binding to rat neurokinin receptors andthus are potential agonists or antagonists of neurokinin receptors. Themethod involves transfecting COS cells with expression vectorscontaining rat neurokinin receptors, allowing the transfected cells togrow for a time sufficient to allow the neurokinin receptors to beexpressed, harvesting the transfected cells and resuspending the cellsin assay buffer containing a known radioactively labeled agonist of theneurokinin receptors either in the presence or the absence of thesubstance, and then measuring the binding of the radioactively labeledknown agonist of the neurokinin receptor to the neurokinin receptor. Ifthe amount of binding of the known agonist is less in the presence ofthe substance than in the absence of the substance, then the substanceis a potential agonist or antagonist of the neurokinin receptor. Wherebinding of the substance such as an agonist or antagonist to ismeasured, such binding can be measured by employing a labeled substanceor agonist. The substance or agonist can be labeled in any convenientmanner known to the art, e.g., radioactively, fluorescently,enzymatically.

Therefore, the specificity of binding of compounds having affinity forSaGluCl shown by measuring the affinity of the compounds for recombinantcells expressing the cloned receptor or for membranes from these cells.Expression of the cloned receptor and screening for compounds that bindto SaGluCl or that inhibit the binding of a known, radiolabeled ligandof SaGluCl (such as glutamate, ivermectin or nodulasporic acid) to thesecells, or membranes prepared from these cells, provides an effectivemethod for the rapid selection of compounds with high affinity forSaGluCl. Such ligands need not necessarily be radiolabeled but can alsobe nonisotopic compounds that can be used to displace bound radiolabeledcompounds or that can be used as activators in functional assays.Compounds identified by the above method again are likely to be agonistsor antagonists of SaGluCl and may be peptides, proteins, ornon-proteinaceous organic molecules. As noted elsewhere in thisspecification, compounds may modulate by increasing or attenuating theexpression of DNA or RNA encoding SaGluCl, or by acting as an agonist orantagonist of the SaGluCl receptor protein. Again, these compounds thatmodulate the expression of DNA or RNA encoding SaGluCl or the biologicalfunction thereof may be detected by a variety of assays. The assay maybe a simple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample.

To this end, the present invention relates in part to methods ofidentifying a substance which modulates SaGluCl1 and/or SaGluCl2receptor activity, which involves:

(a) combining a test substance in the presence and absence of a SaGluCl1and/or SaGluCl2 receptor protein wherein said receptor protein comprisesthe amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8 and/or 10and,

(b) measuring and comparing the effect of the test substance in thepresence and absence of the SaGluCl1 and/or SaGluCl2 receptor protein.

In addition, several specific embodiments are disclosed herein to showthe diverse type of screening or selection assay which the skilledartisan may utilize in tandem with expression vectors directing theexpression of the SaGluCl1 and/or SaGluCl2 receptor protein and anadditional SaGluCl. Methods for identifying agonists and antagonists ofother receptors are well known in the art and can be adapted to identifyagonists and antagonists of SaGluCl1 and/or SaGluCl2. Therefore, theseembodiments are presented as examples and not as limitations. To thisend, the present invention includes assays by which SaGluCl and/orSaGluCl2 modulators (such as agonists and antagonists) may beidentified. Accordingly, the present invention includes a method fordetermining whether a substance is a potential agonist or antagonist ofSaGluCl1 and/or SaGluCl2 that comprises:

(a) transfecting or transforming cells with an expression vector thatdirects expression of SaGluCl and/or SaGluCl2 in the cells;

(b) transfecting or transforming said cells with a second expressionvector that directs expression of a known GluCl subunit whichco-assembles with SaGluCl1 and/or SaGluCl2 resulting in test cells;

(c) allowing the test cells to grow for a time sufficient to allowSaGluCl1 and/or SaGluCl2 to be expressed and for a functional channel tobe generated;

(d) exposing the cells to a labeled ligand of SaGluCl1 and/or SaGluCl2in the presence and in the absence of the substance;

(e) measuring the binding of the labeled ligand to the SaGluCl and/orSaGluCl2 channel; where if the amount of binding of the labeled ligandis less in the presence of the substance than in the absence of thesubstance, then the substance is a potential agonist or antagonist ofSaGluCl1 and/or SaGluCl2.

The conditions under which step (d) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells may be harvested and resuspended in the presence of thesubstance and the labeled ligand. In a modification of theabove-described method, step (d) is modified in that the cells are notharvested and resuspended but rather the radioactively labeled knownagonist and the substance are contacted with the cells while the cellsare attached to a substratum, e.g., tissue culture plates.

The present invention also includes a method for determining whether asubstance is capable of binding to SaGluCl1 and/or SaGluCl2, i.e.,whether the substance is a potential agonist or an antagonist ofSaGluCl1 and/or SaGluCl2 channel activation, where the method comprises:

(a) transfecting or transforming cells with an expression vector thatdirects the expression of SaGluCl1 and/or SaGluCl2 in the cells;

(b) transfecting or transforming said cells with a second expressionvector that directs expression of at least one other known GluCl subunitwhich co-assembles with SaGluCl1 and/or SaGluCl2 resulting in testcells;

(c) exposing the test cells to the substance;

(d) measuring the amount of binding of the substance to SaGluCl1 and/orSaGluCl2;

(e) comparing the amount of binding of the substance to SaGluCl1 and/orSaGluCl2 in the test cells with the amount of binding of the substanceto control cells that have not been transfected with SaGluCl1 and/orSaGluCl2;

wherein if the amount of binding of the substance is greater in the testcells as compared to the control cells, the substance is capable ofbinding to SaGluCl1 and/or SaGluCl2. Determining whether the substanceis actually an agonist or antagonist can then be accomplished by the useof functional assays such as, e.g., the assay involving the use ofpromiscuous G-proteins described below.

The conditions under which step (c) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells are harvested and resuspended in the presence of thesubstance.

Expression of SaGluCl DNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being preferred.

Following expression of SaGluCl in a host cell, SaGluCl protein may berecovered to provide SaGluCl protein in active form. Several SaGluClprotein purification procedures are available and suitable for use.Recombinant SaGluCl protein may be purified from cell lysates andextracts by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. In addition, recombinant SaGluClprotein can be separated from other cellular proteins by use of animmunoaffinity column made with monoclonal or polyclonal antibodiesspecific for full-length SaGluCl protein, or polypeptide fragments ofSaGluCl protein.

Polyclonal or monoclonal antibodies may be raised against SaGluCl or asynthetic peptide (usually from about 9 to about 25 amino acids inlength) from a portion of SaGluCl1 or SaGluCl2 as disclosed in SEQ DNOs:2, 4, 6, 8 and/or 10. Monospecific antibodies to SaGluCl arepurified from mammalian antisera containing antibodies reactive againstSaGluCl or are prepared as monoclonal antibodies reactive with SaGluClusing the technique of Kohler and Milstein (1975, Nature 256: 495–497).Monospecific antibody as used herein is defined as a single antibodyspecies or multiple antibody species with homogenous bindingcharacteristics for SaGluCl. Homogenous binding as used herein refers tothe ability of the antibody species to bind to a specific antigen orepitope, such as those associated with SaGluCl, as described above.Human SaGluCl-specific antibodies are raised by immunizing animals suchas mice, rats, guinea pigs, rabbits, goats, horses and the like, with anappropriate concentration of SaGluCl protein or a synthetic peptidegenerated from a portion of SaGluCl with or without an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 mg and about 1000 mg of SaGluClprotein associated with an acceptable immune adjuvant. Such acceptableadjuvants include, but are not limited to, Freund's complete, Freund'sincomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The initial immunization consists ofSaGluCl protein or peptide fragment thereof in, preferably, Freund'scomplete adjuvant at multiple sites either subcutaneously (SC),intraperitoneally (IP) or both. Each animal is bled at regularintervals, preferably weekly, to determine antibody titer. The animalsmay or may not receive booster injections following the initialimmunization. Those animals receiving booster injections are generallygiven an equal amount of SaGluCl in Freund's incomplete adjuvant by thesame route. Booster injections are given at about three week intervalsuntil maximal titers are obtained. At about 7 days after each boosterimmunization or about weekly after a single immunization, the animalsare bled, the serum collected, and aliquots are stored at about −20° C.

Monoclonal antibodies (mAb) reactive with SaGluCl are prepared byimmunizing inbred mice, preferably Balb/c, with SaGluCl protein. Themice are immunized by the IP or SC route with about 1 mg to about 100mg, preferably about 10 mg, of SaGluCl protein in about 0.5 ml buffer orsaline incorporated in an equal volume of an acceptable adjuvant, asdiscussed above. Freund's complete adjuvant is preferred. The micereceive an initial immunization on day 0 and are rested for about 3 toabout 30 weeks. Immunized mice are given one or more boosterimmunizations of about 1 to about 100 mg of SaGluCl in a buffer solutionsuch as phosphate buffered saline by the intravenous (IV) route.Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions which will allow theformation of stable hybridomas. Fusion partners may include, but are notlimited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, withSp 2/0 being preferred. The antibody producing cells and myeloma cellsare fused in polyethylene glycol, about 1000 mol. wt., at concentrationsfrom about 30% to about 50%. Fused hybridoma cells are selected bygrowth in hypoxanthine, thymidine and aminopterin supplementedDulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.Supernatant fluids are collected form growth positive wells on aboutdays 14, 18, and 21 and are screened for antibody production by animmunoassay such as solid phase immunoradioassay (SPIRA) using SaGluClas the antigen. The culture fluids are also tested in the Ouchterlonyprecipitation assay to determine the isotype of the mAb. Hybridoma cellsfrom antibody positive wells are cloned by a technique such as the softagar technique of MacPherson, 1973, Soft Agar Techniques, in TissueCulture Methods and Applications, Kruse and Paterson, Eds., AcademicPress.

Monoclonal antibodies are produced in vivo by injection of pristineprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8–12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-SaGluCl mAb is carried out by growing thehybridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific mAb. The mAb are purified bytechniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of SaGluCl inbody fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for SaGluCl peptide fragments, or arespective full-length SaGluCl.

SaGluCl antibody affinity columns are made, for example, by adding theantibodies to Affigel-10 (Biorad), a gel support which is pre-activatedwith N-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23 M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) and the cellculture supernatants or cell extracts containing full-length SaGluCl orSaGluCl protein fragments are slowly passed through the column. Thecolumn is then washed with phosphate buffered saline until the opticaldensity (A₂₈₀) falls to background, then the protein is eluted with 0.23M glycine-HCl (pH 2.6). The purified SaGluCl protein is then dialyzedagainst phosphate buffered saline.

The present invention also relates to a non-human transgenic animalwhich is useful for studying the ability of a variety of compounds toact as modulators of either SaGluCl1, SaGluCl2, or any alternativefunctional SaGluCl channel in vivo by providing cells for culture, invitro. In reference to the transgenic animals of this invention,reference is made to transgenes and genes. As used herein, a transgeneis a genetic construct including a gene. The transgene is integratedinto one or more chromosomes in the cells in an animal by methods knownin the art (e.g., U.S. Pat. No. 5,612,205; U.S. Pat. No. 5,721,367, U.S.Pat. No. 5,464,764, U.S. Pat. No. 5,487,992, U.S. Pat. No. 5,627,059,U.S. Pat. No. 5,631,153). Once integrated, the transgene is carried inat least one place in the chromosomes of a transgenic animal. Of course,a gene is a nucleotide sequence that encodes a protein, such as one or acombination of the cDNA clones described herein. The gene and/ortransgene may also include genetic regulatory elements and/or structuralelements known in the art. A type of target cell for transgeneintroduction is the embryonic stem cell (ES). ES cells can be obtainedfrom pre-implantation embryos cultured in vitro and fused with embryos(Evans et al., 1981, Nature 292:154–156; Bradley et al., 1984, Nature309:255–258; Gossler et al., 1986, Proc. Natl. Acad. Sci. USA83:9065–9069; and Robertson et al., 1986 Nature 322:445–448). Transgenescan be efficiently introduced into the ES cells by a variety of standardtechniques such as DNA transfection, microinjection, or byretrovirus-mediated transduction. The resultant transformed ES cells canthereafter be combined with blastocysts from a non-human animal. Theintroduced ES cells thereafter colonize the embryo and contribute to thegerm line of the resulting chimeric animal (Jaenisch, 1988, Science 240:1468–1474). It will also be within the purview of the skilled artisan toproduce transgenic or knock-out ivertebrate animals (e.g., C. elegans)which express the SaGluCl2 transgene in a wild type C. elegans GluClbackground as well in C. elegans mutants knocked out for one or both ofthe C. elegans GluCl subunits. These organisms will be helpful infurther determining the dominant negative effect of SaGluCl2 as well asselecting from compounds which modulate this effect.

Pharmaceutically useful compositions comprising modulators of SaGluClmay be formulated according to known methods such as by the admixture ofa pharmaceutically acceptable carrier. Examples of such carriers andmethods of formulation may be found in Remington's PharmaceuticalSciences. To form a pharmaceutically acceptable composition suitable foreffective administration, such compositions will contain an effectiveamount of the protein, DNA, RNA, modified SaGluCl, or either SaGluClagonists or antagonists including tyrosine kinase activators orinhibitors.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders.The effective amount may vary according to a variety of factors such asthe individual's condition, weight, sex and age. Other factors includethe mode of administration.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages. Alternatively, co-administration orsequential administration of other agents may be desirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds identified according to this inventionas the active ingredient can be administered in a wide variety oftherapeutic dosage forms in conventional vehicles for administration.For example, the compounds can be administered in such oral dosage formsas tablets, capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they mayalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

Advantageously, compounds of the present invention may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three or four times daily. Furthermore, compoundsfor the present invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using those forms of transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal, hepatic and cardiovascular function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

The following examples are provided to illustrate the present inventionwithout, however, limiting the same hereto.

EXAMPLE 1 Isolation and Characterization of DNA Molecules EncodingSaGluCl

The molecular procedures were performed following standard procedureswell known in the art available in references such as Ausubel et. al.(1992, Short protocols in molecular biology. F. M. Ausubel et al.,—2^(nd). ed. (John Wiley & Sons)) and Sambrook et al. (1989, Molecularcloning. A laboratory manual. J. Sambrook, E. F. Fritsch, and T.Maniatis—2^(nd) ed. (Cold Spring Harbor Laboratory Press)). RNA wasisolated from neurons dissected out of grasshopper ventral ganglia,using TRI-REAGENT (Molec. Res. Inc.). First strand cDNA was synthesizedfrom 200 ng RNA using a SUPERSCRIPT preamplification System (LifeTechnologies). A tenth of the first strand reaction was used for PCR.The degenerate oligos utilized were designed based on sequences obtainedfrom C. elegans, Drosophila, and Flea (C. felis) GluCls:

The oligonucleotides used are as follows:

Forward (Oligo 27F2):

GGAT(G/T)CCNGA(C/T)N(C/T)NTT(C/T)TTNN(A/C)NA(A/C)(C/T)G (SEQ ID NO:11)

Reverse 1 (Oligo 3B):

CNA(A/G)(A/C)A(A/G)NGCNC(A/C)GAANA(C/T)(A/G)AA(C/T)G (SEQ ID NO:12)

Reverse 2 (Oligo 3A):

CAN(A/G)CNCCN(A/G)(G/T)CCANAC(A/G)TCNA(C/T)N(A/G)C (SEQ ID NO:13)

Two PCR rounds, using the combinations 27F2+3A, then 27F2+3B″ wereperformed.

The cycles were as follow: 1×(95° C. for 120 sec.), then 30×(95° C. for45 sec.; 50° C. for 90 sec.; and 72° C. for 120 sec.), then 1×(72° C.for 120 sec.). Reagents were from Life Technology Inc. The oligos'concentration was 5 μM. One tenth of the PCR reaction products wastested by Southern blot analysis, in order to identify and prevent thePCR-cloning of contaminating sequence from GluCl clones already in usein the laboratory. Novel PCR products of the appropriate size werecloned into the PCR2.1 plasmid vector using a “TA” cloning kit(Invitrogen, Inc.). Following sequence analysis (ABI Prism, PE AppliedBiosystems), selected PCR clone inserts were radiolabelled and used asprobes to screen a cDNA library generated into the Uni-ZAP vector(Stratagene, Inc.) from using and a poly (A) enriched fraction from theRNA mentioned above. Sequences obtained from full-length cDNA clones(ABI Prism, PE Applied Biosystems) were analyzed using the GCG Inc.package. Subcloning of SaGluCl1 and of SaGluCl2 into a mammalianexpression vector was done by excision of whole inserts (EcoRI+XhoIexcision) from the UniZap pBS plasmid, followed by ligation into theTetSplice (Cat. No. 10583-011; Life Technologies/GIBCO BRL). FIGS. 1A–1Fand FIGS. 2A–2B show the derived nucleotide and amino acid sequencecovering the open reading frames for SaGluCl1 and SaGluCl2 cDNA clones.

EXAMPLE 2 Functional Eexpression of GluCls Clones in Xenopus Oocytes

Full length cDNA clones corresponding to the selected RT-PCR sequenceswere used as template for synthesis of in vitro transcribed RNA (AmbionInc.). The full-length cDNA encoding SaGluCl1 in a Bluescript plasmid islinearized and capped cRNA transcripts are synthesized using appropriateoligonucleotide primers and the mMESSAGE mMACHINE in vitro RNAtranscription kit from Ambion. Xenopus laevis oocytes were prepared andinjected using standard methods as described (Arena et al., 1991, Mol.Pharmacol. 40: 368–374; Arena et al, 1992, Mol. Brain Res. 15: 339–348).Adult female Xenopus laevis were anesthetized with 0.17% tricainemethanesulfonate and the ovaries were surgically removed and placed in adish consisting of (mM): NaCl 82.5, KCl 2, MgCl₂ 1, CaCl₂ 1.8, HEPES 5,adjusted to pH 7.5 with NaOH(OR-2). Ovarian lobes were broken open,rinsed several times, and gently shaken in OR-2 containing 0.2%collagenase (Sigma, Type 1A) for 2–5 hours. When approximately 50% ofthe follicular layers were removed, Stage V and VI oocytes were selectedand placed in media consisting of (mM): NaCl 86, KCl 2, MgCl₂ 1, CaCl₂1.8, HEPES 5, Na pyruvate 2.5, theophylline 0.5, gentamicin 0.1 adjustedto pH 7.5 with NaOH (ND-96) for 24–48 hours before injection. For mostexperiments, oocytes were injected with 10 ng of cRNA in 50 nl of RNasefree water. Control oocytes were injected with 50 nl of water. Oocyteswere incubated for 1–5 days in ND-96 supplemented with 50 mg/mlgentamycin, 2.5 mM Na pyruvate and 0.5 mM theophylline before recording.Incubations and collagenase digestion were carried out at 18° C.

Voltage-clamp studies were conducted with the two microelectrode voltageclamp technique using a Dagan CA1 amplifier (Dagan Instruments,Minneapolis, Minn.). The current passing microelectrodes were filledwith 0.7 M KCl plus 1.7 M K₃-citrate and the voltage recordingmicroelectrodes were filled with 1.0 M KCl. The extracellular solutionfor most experiments was saline consisting of (mM): NaCl 96, BaCl₂ 3.5,MgCl₂ 0.5, CaCl₂ 0.1, HEPES 5, adjusted to pH 7.5 with NaOH. Theextracellular chloride concentration was reduced in some experiments byequimolar replacement of NaCl with the sodium salt of the indicatedanion. Experiments were conducted at 21–24° C. Data were acquired usingthe program Pulse and most analysis was performed with the companionprogram Pulsefit (Instrutech Instruments, Great Neck, N.Y.) or with IgorPro (Wavemetrics, Lake Oswego, Oreg.). Data were filtered (f_(c), −3 db)at 1 kHz, unless otherwise indicated. FIG. 3 shows the results of theexperiment in which the clone SaGluCl1 (short form “SC”) was expressedin a Xenopus oocyte. The measurement was made as described in thisExample with the two microelectrode voltage clamp technique and themembrane potential was held at 0 mV. Two current recordings aresuperimposed. The bars at top show the duration of application ofglutamate and ivermectin phosphate. Glutamate was applied first andelicited a rapidly desensitizing current. Ivermectin phosphate eliciteda current of similar amplitude, but the channel stayed open much longer.This indicates that expression of this protein reconstitutes afunctional ion channel that responds to both glutamate and ivermectin.

EXAMPLE 3 Functional Expression of GluCls Clones in Mammalian Cells

PTet-Splice subclones of SaGluCl1 and SaGluCl2 as disclosed in Examplesection 1 were transfected into CHO Tet-Off cells (Clontech labs, Inc.)using the Lipofectamine Plus reagent (Life Technologies Inc.). Stablecell lines are selected by growth in the presence of G418. Single G418resistant clones are isolated and shown to contain the intact SaGluClgene. Clones containing the SaGluCl cDNAs may be analyzed for expressionusing immunological techniques, such as immuneprecipitation, Westernblot, and immunofluorescence using antibodies specific to the SaGluClproteins. Antibody may obtained from rabbits innoculated with peptidesthat are synthesized from the amino acid sequence predicted from theSaGluCl sequences. Expression is also analyzed using patch clampelectrophysiological techniques, an anion flux assay, and ³H-ivermectinand ³H-glutamate binding assays.

Cells that are expressing SaGluCl stably or transiently, are used totest for expression of avermectin, glutamate, sensitive chloridechannels and for ligand binding activity. These cells are used toidentify and examine other compounds for their ability to modulate,inhibit or activate the avermectin, glutamate sensitive chloride channeland to compete for binding with radioactive avermectin, glutamate,derivatives. These cells are used to identify and examine othercompounds which modulate SaGluCl activity with an anion flux assay.

Cassettes containing the SaGluCl cDNA in the positive orientation withrespect to the promoter are ligated into appropriate restriction sites3′ of the promoter and identified by restriction site mapping and/orsequencing. These cDNA expression vectors are introduced into host cellsfor, example CHO cells, COS-7 (ATCC# CRL1651), and CV-1 tat [Sackevitzet al., Science 238: 1575 (1987)], 293, L (ATCC# CRL6362)] by standardmethods including but not limited to electroporation, or chemicalprocedures (cationic liposomes, DEAE dextran, calcium phosphate).Transfected cells and cell culture supernatants can be harvested andanalyzed for SaGluCl expression.

All of the vectors used for mammalian transient expression can be usedto establish stable cell lines expressing SaGluCl. Unaltered SaGluClcDNA constructs cloned into expression vectors are expected to programhost cells to make SaGluCl protein. In addition, SaGluCl is expressedextracellularly as a secreted protein by ligating SaGluCl GluCl cDNAconstructs to DNA encoding the signal sequence of a secreted protein.The transfection host cells include, but are not limited to, CV-1-P[Sackevitz et al., Science 238: 1575 (1987)], tk-L [Wigler, et al., Cell11: 223 (1977)], NS/0, and dHFr-CHO [Kaufman and Sharp, J. Mol. Biol.159: 601, (1982)].

Co-transfection of any vector containing SaGluCl GluCl with a drugselection plasmid including, but not limited to G418, aminoglycosidephosphotransferase; hygromycin, hygromycin-B phosphotransferase; APRT,xanthine-guanine phosphoribosyl-transferase, will allow for theselection of stably transfected clones. Levels of SaGluCl GluCl arequantitated by the assays described herein.

SaGluCl cDNA constructs are also ligated into vectors containingamplifiable drug-resistance markers for the production of mammalian cellclones synthesizing the highest possible levels of SaGluCl. Followingintroduction of these constructs into cells, clones containing theplasmid are selected with the appropriate agent, and isolation of anover-expressing clone with a high copy number of plasmids isaccomplished by selection with increasing doses of the agent.

The expression of recombinant SaGluCl1 and/or SaGluCl2 is achieved bytransfection of the full-length SaGluCl cDNA into a mammalian host celldescribed herein. Functional expression of SaGluCl1 in CHO cells isshown in FIGS. 4A and 4B, which indicates that this subunit can assembleinto a homomultimer channel activated by ivermectin (IVM, FIG. 4A) andnodulasporic acid (NA, FIG. 4B).

EXAMPLE 4 Cloning of SaGluCl GluCl cDNA into a Baculovirus ExpressionVector for Expression in Insect Cells

Baculovirus vectors, which are derived from the genome of the AcNPVvirus, are designed to provide high level expression of cDNA in the Sf9line of insect cells (ATCC CRL# 1711). A recombinant baculoviruseexpressing SaGluCl cDNA is produced by the following standard methods(In Vitrogen Maxbac Manual): the SaGluCl cDNA constructs are ligatedinto the polyhedrin gene in a variety of baculovirus transfer vectors,including the pAC360 and the BlueBac vector (In Vitrogen). Recombinantbaculoviruses are generated by homologous recombination followingco-transfection of the baculovirus transfer vector and linearized AcNPVgenomic DNA [Kitts, 1990, Nuc. Acid. Res. 18: 5667] into Sf9-cells.Recombinant pAC360 viruses are identified by the absence of inclusionbodies in infected cells and recombinant pBlueBac viruses are identifiedon the basis of b-galactosidase expression (Summers, M. D. and Smith, G.E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaquepurification, SaGluCl expression is measured by the assays describedherein.

The cDNA encoding the entire open reading frame for SaGluCl GluCl isinserted into the BamHI site of pBlueBacII. Constructs in the positiveorientation are identified by sequence analysis and used to transfectSf9 cells in the presence of linear AcNPV mild type DNA.

Authentic, active SaGluCl is found in the cytoplasm of infected cells.Active SaGluCl is extracted from infected cells by hypotonic ordetergent lysis.

EXAMPLE 5 Cloning of SaGluCl GluCl cDNA into a Yeast Expression Vector

Recombinant SaGluCl is produced in the yeast S. cerevisiae following theinsertion of the optimal SaGluCl cDNA cistron into expression vectorsdesigned to direct the intracellular or extracellular expression ofheterologous proteins. In the case of intracellular expression, vectorssuch as EmBLyex4 or the like are ligated to the SaGluCl cistron [Rinas,et al., 1990, Biotechnology 8: 543–545; Horowitz B. et al., 1989, J.Biol. Chem. 265: 4189–4192]. For extracellular expression, the SaGluClGluCl cistron is ligated into yeast expression vectors which fuse asecretion signal (a yeast or mammalian peptide) to the NH₂ terminus ofthe SaGluCl protein [Jacobson, 1989, Gene 85: 511–516; Riett and Bellon,1989, Biochem. 28: 2941–2949].

These vectors include, but are not limited to pAVE1-6, which fuses thehuman serum albumin signal to the expressed cDNA [Steep, 1990,Biotechnology 8: 42–46], and the vector pL8PL which fuses the humanlysozyme signal to the expressed cDNA [Yamamoto, Biochem. 28:2728–2732)]. In addition, SaGluCl is expressed in yeast as a fusionprotein conjugated to ubiquitin utilizing the vector pVEP [Ecker, 1989,J. Biol. Chem. 264: 7715–7719, Sabin, 1989 Biotechnology 7: 705–709,McDonnell, 1989, Mol. Cell Biol. 9: 5517–5523 (1989)]. The levels ofexpressed SaGluCl are determined by the assays described herein.

EXAMPLE 6 Purification of Recombinant SaGluCl CluCl

Recombinantly produced SaGluCl may be purified by antibody affinitychromatography. SaGluCl GluCl antibody affinity columns are made byadding the anti-SaGluCl GluCl antibodies to Affigel-10 (Biorad), a gelsupport which is pre-activated with N-hydroxysuccinimide esters suchthat the antibodies form covalent linkages with the agarose gel beadsupport. The antibodies are then coupled to the gel via amide bonds withthe spacer arm. The remaining activated esters are then quenched with 1Methanolamine HCl (pH 8). The column is washed with water followed by0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody orextraneous protein. The column is then equilibrated in phosphatebuffered saline (pH 7.3) together with appropriate membrane solubilizingagents such as detergents and the cell culture supernatants or cellextracts containing solubilized SaGluCl are slowly passed through thecolumn. The column is then washed with phosphate-buffered salinetogether with detergents until the optical density (A280) falls tobackground, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6)together with detergents. The purified SaGluCl protein is then dialyzedagainst phosphate buffered saline.

1. A purified DNA molecule encoding a S. americana GluCl1 channelprotein which comprises an amino acid sequence of SEQ ID NO:8.
 2. Anexpression vector for expressing a S. americana GluCl1 channel proteinin a recombinant host cell wherein said expression vector comprises theDNA molecule of claim
 1. 3. An isolated host cell which expresses arecombinant S. americana GluCl1 channel protein wherein said host cellcontains the expression vector of claim
 2. 4. A process for expressing aS. americana GluCl1 channel protein in a recombinant host cell,comprising: (a) transfecting the expression vector of claim 2 into asuitable host cell; and, (b) culturing the host cell of step (a) underconditions which allow expression of said S. americana GluCl channelprotein from said expression vector.
 5. A purified DNA molecule encodinga S. americana GluCl1 channel protein which consists of an amino acidsequence as set forth in SEQ ID NO:8.
 6. An expression vector forexpressing a S. americana GluCl1 channel protein in a recombinant hostcell wherein said expression vector comprises the DNA molecule of claim5.
 7. An isolated host cell which expresses a recombinant S. americanaGluCl1 channel protein wherein said host cell contains the expressionvector of claim
 6. 8. A process for expressing a S. americana GluCl1channel protein in a recombinant host cell, comprising: (a) transfectingthe expression vector of claim 6 into a suitable host cell; and, (b)culturing the host cell of step (a) under conditions which allowexpression of said S. americana GluCl channel protein from saidexpression vector.
 9. A purified DNA molecule encoding a recombinant S.americana GluCl1 channel protein wherein said DNA molecule comprises anucleotide sequence of SEQ ID NO:7.
 10. An expression vector forexpressing a recombinant S. americana GluCl1 channel protein whereinsaid expression vector comprises the DNA molecule of claim
 9. 11. Anisolated host cell which expresses a recombinant S. americana GluCl1channel protein wherein said host cell contains the expression vector ofclaim
 10. 12. A process for expressing a recombinant S. americana GluCl1channel protein in a recombinant host cell, comprising: (a) transfectingthe expression vector of claim 10 into a suitable host cell; and, (b)culturing the host cell of step (a) under conditions which allowexpression of said recombinant S. Americana GluCl1 channel protein fromsaid expression vector.
 13. An isolated S. americana GluCl1 channelprotein which comprises an amino acid sequence of SEQ ID NO:8.
 14. Theisolated S. americana GluCl1 channel protein of claim 13 which is aproduct of a DNA expression vector contained within an isolatedrecombinant host cell.
 15. A substantially pure membrane preparationcomprising the S. americana GluCl1 channel protein purified from theisolated recombinant host cell of claim
 14. 16. An isolated S. americanaGluCl1 channel protein which consists of the amino acid sequence of SEQID NO:8.
 17. The isolated S. americana GluCl1 channel protein of claim16 which is a product of a DNA expression vector contained within anisolated recombinant host cell.
 18. A substantially pure membranepreparation comprising the S. americana GluCl1 channel protein purifiedfrom the recombinant host cell of claim 17.